Lysosomal enzyme-cleavable antitumor drug conjugates

ABSTRACT

The present invention relates to drug-ligand conjugates wherein the drug is linked to the ligand through a protein peptide linker and a connector, a process for the preparation of the conjugates, method of controlling the growth of undesirable cells, pharmaceutical compositions, and intermediates thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides drug-ligand conjugates wherein the ligandis connected to the drug moiety through a peptide linker made up of aprotein peptide specifier, a carboxylic acyl unit, and a self-immolatingspacer, and which conjugates are activated by lysosomal enzymes.

2. Description of the Art

Bipartate compounds consisting of a carrier or linker moiety and a drugmoiety are known. These compounds have been particularly useful in theformation of immunoconjugates directed against tumor associatedantigens. In certain cases, however, bipartate compounds may be unstabledue to the inherent nature of the bond linking the antibodies to thedrug or due to the electronic or steric features of the drug moietywhich may hinder hydrolysis of the bond by the desired target enzyme.Katzenellenbogen, J. Amer. Chem. Soc., (1981) 24: 479-480.

SUMMARY OF THE INVENTION

The present invention provides tumor specific, drug-ligand conjugatescomposed of a ligand, a drug, and a peptide linker, which conjugate isselectively activatible at the site of the tumor.

The drug-ligand conjugates of this invention comprise at least one drugmolecule, a ligand capable of targeting a selected cell population, anda peptide linker which contains a carboxylic acyl, and a protein peptidespecifier. The peptide linker may also contain a self-immolating spacerwhich spaces the protein peptide sequence and the drug.

The ligand is linked to the carboxylic acyl unit via athioether-containing linker unit arm, which thioether bond is created byreaction of a sulfhydryl group on the ligand. In a preferred embodiment,the targeting ligand is attached directly to the peptide linker througha covalent thioether bond.

An aspect of the present invention provides drug conjugates which areselectively activatible at the site of the tumor.

Another aspect of the invention provides tumor-specific drug conjugateswhich are highly selective substrates for drug-activating enzymaticcleavage by one or more tumor-associated enzymes.

A further aspect of the invention provides tumor-specific drugconjugates wherein the activating enzyme is one which is present in thetumor in sufficient amounts to generate cytotoxic levels of free drug inthe vicinity of the tumor.

Another aspect of the invention provides tumor-specific drug conjugateswhich tumor specificity arises solely from the ligand.

Another aspect of the invention provides tumor-specific drug conjugateswhich are stable to adventitious proteases in blood.

A still further aspect of the present invention provides atumor-specific drug conjugate in accordance with the preceding aspects,which is considerably less toxic than the activated drug.

In another aspect the present invention provides a method for theproduction of the drug conjugates and pharmaceutical compositions andmethods for delivering the conjugates to target cells in which amodification in biological process is desired, such as in the treatmentof diseases such as cancer.

The present invention also provides a method for delivering to the siteof tumor cells in a warm-blooded animal an active antitumor drug byadministering to said warm-blooded animal the drug-ligand conjugateaccording to this invention.

In one embodiment the drug moiety is an anthracycline antibiotic, theligand is an antibody, A is a carboxylic acyl unit, Y is Phe, Z is Lys,and n is 5.

In a preferred embodiments the anthracycline drug moiety is doxorubicin,the ligand moiety is a chimeric antibody, A is carboxylic acyl unit, Yis Phe, Z is Lys, and n is 5.

In another preferred embodiment the drug moiety is taxol, the ligand isan antibody, Y is Phe, Z is Lys and n is 5.

In another preferred embodiment the drug moiety is mitomycin C, theligand is an antibody, Y is Phe, Z is Lys and n is 5.

The above and other aspects of the present invention are achieved byderivatizing an antitumor agent linked to a ligand through a peptidelinker, made up of a protein peptide sequence and a self-immolatingspacer, at a reactive site appropriate for inhibiting thepharmacological activity of the antitumor agent to thereby convert theantitumor agent into a pharmacologically inactive peptidyl derivativeconjugate. The peptide linker has an amino acid residue sequencespecifically tailored so as to render the peptidyl derivative aselective substrate for drug-activating enzymatic cleavage by one ormore lysosomal proteases, such as cathepsin B, C or D. The enzymaticcleavage reaction will remove the peptide linker moiety from the drugconjugate and effect release of the antitumor agent in pharmacologicallyactive form selectively at the tumor site. In comparison withligand-drug linkers which rely on simple acid hydrolysis for drugrelease this new method provides significantly less systemic toxicitydue to premature linker hydrolysis in the blood, consequently a greateramount of drug is delivered to the tumor site, and the method results ina longer storage life and simplified handling conditions for theconjugate.

The drug-ligand conjugates of the present invention show significantlyless systemic toxicity than biparte conjugates and free drug. Theconjugates of the invention retain both specificity and therapeutic drugactivity for the treatment of a selected target cell population. Theymay be used in a pharmaceutical composition, such as one comprising apharmaceutically effective amount of a compound of Formula (I) below,associated with a pharmaceutically acceptable carrier, diluent orexcipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the expression of the BR96 antigen in theL2987 lung line.

FIGS. 1C and 1D illustrate the expression of the BR96 antigen in theA2780 ovarian line.

FIGS. 1E and 1F illustrate expression of the BR96 antigen in the HCT116colon line.

FIG. 2A shows the potency of the BR96-doxorubicin conjugate and theunconjugated doxorubicin in the L2987 lung line.

FIG. 2B shows the potency of the BR96-doxorubicin conjugate and theunconjugated doxorubicin in the A2780 ovarian line.

FIG. 2C shows the potency of the BR96-doxorubicin conjugate and theunconjugated doxorubicin in the HCT116 colon line.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is provided so that the invention maybe more fully understood.

The present invention provides novel drug-ligand conjugates composed ofa ligand capable of targeting a selected cell population, and a drugconnected to the ligand by a peptide linker. The peptide linker is madeup of a carboxylic acyl unit and a protein peptide sequence. The peptidelinker may also contain a self-immolating spacer, which spaces the drugand the protein peptide sequence.

The ligand molecule can be an immunoreactive protein such as anantibody, or fragment thereof, a non-immunoreactive protein, or peptideligand such as bombesin or, a binding ligand recognizing a cellassociated receptor such as a lectin, or any protein or peptide thatpossesses a reactive sulfhydryl group (—SH) or can be modified tocontain such a sulfhydryl group. The carboxylic acyl unit is linked tothe ligand via a thioether bond, and the drug is linked to the linkervia a functional group selected from primary or seconday amine,hydroxyl, sulfhydryl, carboxyl, aldehyde or ketone.

A conjugate of the present invention is represented by general Formula(I):

in which

D is a drug moiety;

L is a ligand;

A is a carboxylic acyl unit

Y is an amino acid;

Z is an amino acid;

X is a self-immolative spacer;

W is a self-immolative spacer;

m is an integer of 1, 2, 3, 4, 5 or 6.

n is an integer of 0 or 1.

For a better understanding of the invention, the drugs, ligands,peptides and spacers will be discussed individually. The synthesis ofthe conjugates then will be explained.

It will be understood that in the following detailed description andappended claims, the abbreviations and nomenclature employed are thosewhich are standard in amino acid and peptide chemistry, and that all theamino acids referred to are in the L-form unless otherwise specified.

The abbreviations used in the present application, unless otherwiseindicated are as follows:

AcOH: acetic acid; Ala: L-alanine; Alloc: allyloxy-carbonyl; Arg:L-arginine; Boc: t-butyloxycarbonyl; Cit: L-citrulline; DBU:diazobicycloundecene; DCC: dicyclohexylcarbodiimide; DCI: directchemical ionization; DCU: dicyclohexylurea; DIEA: diisopropylethylamine;DMAP: 4-dimethylaminopyridine; DME: 1,2-dimethoxyethane; DOX:doxorubicin; DTT: dithiothreitol; EEDQ:N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; EtOAc: ethyl acetate;FAB: fast atom bombardment; Fmoc: fluorenylmethoxycarbonyl; GABA:γ-aminobutyric acid; Gly: glycine; HOBt: N-hydroxybenzotriazole; HRMS:high resolution mass spectroscopy; LDL: low density lipoprotein; Ile:L-isoleucine; Leu: L-leucine; Lys: L-lysine; MC: 6-maleimidocaproyl;MMA: mitomycin A, MMC: mitomycin C; Mtr: 4-methoxytrityl; NHS:N-hydroxysuccinimide; NMP: N-methylpyrrolidinone; PABC:p-aminobenzyl-carbamoyl; PAB-OH: p-aminobenzyl alcohol;Phe:L-phenylalanine; PNP: p-nitrophenol; TFA: trifluoroacetic acid; THF:tetrahydrofuran; Trp:L-tryptophan; Val: L-valine; Z: benzyloxycarbonyl.

THE PEPTIDE LINKER

The peptide linker of the present invention is made up of a carboxylicacyl unit, and a protein peptide sequence. The linker may also contain aself-immolating spacer which spaces the drug and the protein peptidesequence.

In the conjugate of Formula I, the peptide linker is represented by“A—Y—Z—X—W” in which “A” is the carboxylic acyl unit, “Y” and “Z” areeach amino acids and together form the protein peptide sequence, and “X”and “W” are individualy self-immolating spacers which spaces the proteinpeptide and the drug.

THE PROTEIN PEPTIDE SEQUENCE

In the conjugate of Formula I,

Y is at least one amino acid selected from the group consisting ofalanine, valine, leucine, isoleucine, methionine, phenylalanine,tryptophan and proline, preferably phenylalanine or valine; and

Z is at least one amino acid selected from the group consisting oflysine, lysine protected with acetyl or formyl, arginine, arginineprotected with tosyl or nitro groups, histidine, ornithine, ornithineprotected with acetyl or formyl, and citrulline, preferably lysine, orcitrulline.

The amino acid residue sequence is specifically tailored so that it willbe selectively enzymatically cleaved from the resulting peptidylderivative drug-conjugate by one or more of the tumor-associatedproteases.

The amino acid residue chain length of the peptide linker preferablyranges from that of a dipeptide to that of a tetrapeptide. It will beunderstood, however, that peptide linkers as long as eight amino acidresidues may also suitably be employed.

The following group of exemplary peptide linker groups, are named inorder to illustrate further the conjugates of the present invention:

Phe-Lys, Val-Lys, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys,Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly[SEQ ID NO: 1], Ala-Leu-Ala-Leu [SEQ ID NO:2], Phe-N⁹-tosyl-Arg, andPhe-N⁹-Nitro-Arg.

Specific examples of the preferred embodiment of peptide sequencesinclude Phe-Lys, Val-Lys, Val-Cit, and D-Phe-L-Phe-Lys.

Numerous specific peptide linker molecules suitable for use in thepresent invention can be designed and optimized in their selectivity forenzymatic cleavage by a particular tumor-associated protease. Thepreferred peptide linkers for use in the present invention are thosewhich are optimized toward the proteases, cathepsin B, C and D.Cathepsin B was shown to release DOX from the conjugate at pH 5.3 (37°C.) with (t_(½)=3.0 hrs.).

THE SPACER

The drug-conjugates in accordance with the present invention may employan intermediate self-immolative spacer moiety which spaces andcovalently links together the drug moiety and the protein peptidemoiety. A self-immolative spacer may be defined as a bifunctionalchemical moiety which is capable of covalently linking together twospaced chemical moieties into a normally stable tripartate molecule,releasing one of said spaced chemical moieties from the tripartatemolecule by means of enzymatic cleavage; and following said enzymaticcleavage, spontaneously cleaving from the remainder of the molecule torelease the other of said spaced chemical moieties. In accordance withthe present invention, the self-immolative spacer is covalently linkedat one of its ends to the protein peptide moiety and covalently linkedat its other end to the chemical reactive site of the drug moiety whosederivatization inhibits pharmacological activity, so as to space andcovalently link together the protein peptide moiety and the drug moietyinto a tripartate molecule which is stable and pharmacologicallyinactive in the absence of the target enzyme, but which is enzymaticallycleavable by such target enzyme at the bond covalently linking thespacer moiety and the protein peptide moiety to thereby effect releaseof the protein peptide moiety from the tripartate molecule. Suchenzymatic cleavage, in turn, will activate the self-immolating characterof the spacer moiety and initiate spontaneous cleavage of the bondcovalently linking the spacer moiety to the drug moiety, to therebyeffect release of the drug in pharmacologically active form.

In the conjugate of Formula I,

X is a spacer moiety which spaces and covalently links together the drugmoiety and the amino acid, in which the spacer is linked to the drugmoiety via the T moiety, and which may be represented by the compoundsof Formulae (III), (IV), (V) or (VI):

in which T is O, NH, N or S,

—HN—R¹—COT  Formula (IV)

in which T is O, NH, N or S, and

R¹ is C₁-C₅ alkyl;

(J. Med. Chem., 27: 1447 (1984))

in which T is O, NH, N or S, and

R² is H or C₁-C₅ alkyl,

or

W is a spacer moiety represented by the Formula (VII)

wherein T is O, S or NH, N.

As used herein “C₁-C₅ alkyl” is meant to include branched or unbranchedhydrocarbon chain having, unless otherwise noted, one to five carbonatoms, including but not limited to methyl, ethyl, isopropyl, n-propyl,sec-butyl, isobutyl, n-butyl and the like.

A preferred spacer moiety suitable for use in the present invention isPABC represented by the Formula (IIIa):

Another preferred spacer moiety suitable for use in the presentinvention is GABA represented by the Formula (IVa):

Yet another preferred spacer moiety suitable for use in the presentinvention is α,α-dimethyl GABA represented by the Formula (IVb):

Another preferred spacer moiety suitable for use in the presentinvention is β,β-dimethyl GABA represented by the Formula (IVc):

THE CARBOXYLIC ACYL UNIT

In the conjugate of Formula (I), the carboxylic unit “A” is linked tothe ligand via a sulfur atom derived from the ligand. Representative ofconjugates of this invention are compounds of Formulae (IXa), (IXb),(IXc), (IXd) and (IXe), which “A” is the compound in brackets.

in which q is 1-10, and L, Y, Z, X, W, D, n and m are as previouslydefined;

made from succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(SMCC) (Pierce Catalog p. E-15 (1992)), wherein L, Y, Z, X, W, D, n andm are as previouly defined;

made from m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (PierceCatalog p. E-16 (1992)), wherein L, Y, Z, X, W, D, n and m are aspreviously defined;

made from succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB) (Piercecatalog p. E-18 (1992), wherein L, Y, Z, X, W, D, n and m are aspreviously defined;

made from N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) (Piercecatalog p. E-17 (1992)), wherein L, Y, Z, X, W, D, n and m are aspreviously defined; or

A is a compound that joins up to the peptide and is linked to the ligandvia a sulfur atom derived from the ligand, and a sulfur atom derivedfrom the carboxylic acyl unit to form a dithio link. Representative ofconjugates of this invention are compounds of Formulae (Xa), (Xb) and(Xc)

made from N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Piercecatalog p. E-13 (1992)), wherein L, Y, Z, X, W, D, n and m are aspreviously defined;

made from 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene(SMPT) (Pierce catalog p. E-12 (1992)), wherein L, Y, Z, X, W, D, n andm are as previously defined; and

made from long chain SPDP (Pierce catalog p. E-14 (1992), wherein L, Y,Z, X, W, D, n and m are as previously defined.

THE DRUG

The drug conjugates of the present invention are effective for the usualpurposes for which the corresponding drugs are effective, and havesuperior efficacy because of the ability, inherent in the ligand, totransport the drug to the desired cell where it is of particularbenefit. Further, because the conjugates of the invention can be usedfor modifying a given biological response, the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, aprotein such as tumor necrosis factor.

The preferred drugs for use in the present invention are cytotoxicdrugs, particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Preferred classes of cytotoxic agentsinclude, for example, the enzyme inhibitors such as dihydrofolatereductase inhibitors, and thymidylate synthase inhibitors, DNAintercalators, DNA cleavers, topoisomerase inhibitors, the anthracyclinefamily of drugs, the vinca drugs, the mitomycins, the bleomycins, thecytotoxic nucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, differentiation inducers, and taxols. Particularlyuseful members of those classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, carminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxol, taxotere retinoic acid, butyric acid,N⁸-acetyl spermidine, camptothecin, and their analogues.

As noted previously, one skilled in the art may make chemicalmodifications to the desired compound in order to make reactions of thatcompound more convenient for purposes of preparing conjugates of theinvention.

In the conjugate of Formula I,

D is a drug moiety having pendant to the backbone thereof a chemicallyreactive functional group by means of which the drug backbone is bondedto the protein peptide linker, said functional group selected from thegroup consisting of a primary or secondary amine, hydroxyl, sulfhydryl,carboxyl, aldehyde or a ketone.

Representative of said amino containing drugs are mitomycin-C,mitomycin-A, daunorubicin, doxorubicin, aminopterin, actinomycin,bleomycin, 9-amino camptothecin, N⁸-acetyl spermidine, 1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, cytarabineand derivatives thereof.

Representative of said alcohol group containing drugs are etoposide,camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4-9-diene-2,6-diyne-13-one, (U.S. Pat. No. 5,198,560),podophyllotoxin, anguidine, vincristine, vinblastine,morpholine-doxorubicin, n-(5,5-diacetoxy-pentyl) doxorubicin, andderivatives thereof.

Representative of said sulfhydryl containing drugs are esperamicin and6-mercaptopurine, and derivatives thereof.

Representative of said carboxyl containing drugs are methotrexate,camptothecin (ring-opened form of the lactone), butyric acid, retinoicacid, and derivatives thereof.

Representative of said aldehyde and ketone containing drugs areanguidine and anthracyclines such as doxorubicin, and derivativesthereof.

A highly preferred group of cytotoxic agents for use as drugs in thepresent invention include drugs of the following formulae:

THE MITOMYCIN GROUP OF FORMULA (1):

in which

R¹ is hydrogen or methyl;

R² is —NH₂, —OCH₃, —O(CH₂)₂OH, —NH(CH₂)₂SS(CH₂)₂NHAc, —NHCH—C≡CH,—NH(CH₂)₂SS (C₆H₄)NO₂, —O(CH₂)₂SS(CH₂)₂OH, —N═CH—NHOCH₃, —NH(C₆H₄)OH,—NH(CH₂)₂SS (CH₂)₂NHCO(CH₂)₂CH(NH₂)COOH

THE BLEOMYCIN GROUP OF FORMULA (2):

in which

R¹ is hydroxy, amino, C₁-C₃ alkylamino, di(C₁-C₃ alkyl)amino, C₄-C₆polymethylene amino,

THE METHOTREXATE GROUP OF FORMULA (3):

in which

R¹ is amino or hydroxy;

R² is hydrogen or methyl;

R³ is hydrogen, fluoro, chloro, bromo or iodo;

R⁴ is hydroxy or a moiety which completes a salt of the carboxylic acid.

MELPHALAN OF FORMULA (4):

MERCAPTOPURINE OF FORMULA (5):

A CYTOSINE ARABINOSIDE OF FORMULA (6):

THE PODOPHYLLOTOXINS OF FORMULA (7):

wherein

R² is hydrogen,

R¹ is hydrogen or

wherein

R³ is NH₂, OH, OCH₃, NH(C₁-C₃ alkyl) or

N(C₁-C₃ alkyl)₂

R⁴ is OH, or NH₂,

R⁵ is methyl or thienyl,

R⁶ is hydrogen or methyl, or a phosphate salt thereof.

As used herein “C₁-C₃ alkyl” means a straight or branched carbon chainhaving from one to three carbon atoms; examples include methyl, ethyl,n-propyl and isopropyl.

THE VINCA ALKALOID GROUP OF DRUGS OF FORMULA (8):

in which

R¹ is H, CH₃ or CHO;

when R² and R³ are taken singly, R³ is H, and one of R⁴ and R² is ethyland the other is H or OH;

when R² and R³ are taken together with the carbons to which they areattached, they form an oxirane ring in which case R⁴ is ethyl;

R⁵ is hydrogen, (C₁-C₃ alkyl)—CO, or

chlorosubstituted (C₁-C₃ alkyl)—CO.

As used herein “C₁-C₃ alkyl” means a straight or branched carbon chainhaving from one to three carbon atoms; examples include methyl, ethyl,n-propyl and isopropyl.

DIFLUORONUCLEOSIDES OF FORMULA (9):

in which R¹ is a base of one of the formulae:

in which

R² is hydrogen, methyl, bromo, fluoro, chloro, or iodo;

R³ is —OH or —NH₂;

R⁴ is hydrogen, bromo, chloro, or iodo.

TAXOLS OF FORMULA (10):

wherein

R¹ is hydroxy;

R² is hydrogen or hydroxy;

R²′ is hydrogen or fluoro;

R³ is hydrogen, hydroxy, or acetoxy;

R⁴ is aryl, substituted aryl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl ort-butoxy;

R⁵ is C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, or —Z—R⁶;

Z is a direct bond, C₁₋₆ alkyl or C₂₋₆ alkenyl;

R⁶ is aryl, substituted aryl, C₃₋₆ cycloalkyl, thienyl or furyl.

As used herein, “alkyl” means a straight or branched saturated carbonchain having from one to six carbon atoms; examples include methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl,n-pentyl, sec-pentyl, isopentyl, and n-hexyl. “Alkenyl” means a straightor branched carbon chain having at least one carbon-carbon double bond,and having from two to six carbon atoms; examples include ethenyl,propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, and hexenyl.“Alkynyl” means a straight or branched carbon chain having at least onecarbon-carbon triple bond, and from two to six carbon atoms; examplesinclude ethynyl, propynyl, butynyl, and hexynyl. “Aryl” means aromatichydrocarbon having from six to ten carbon atoms; examples include phenyland naphthyl. “Substituted aryl” means aryl substituted with at leastone group selected from C₁₋₆ alkanoyloxy, hydroxy, halogen, C₁₋₆ alkyl,trifluoromethyl, C₁₋₆ alkoxy, aryl, C₂₋₆ alkenyl, C₁₋₆ alkanoyl, nitro,amino, and amido.

ANGUIDINES OF FORMULA (11):

wherein

R¹ is OH or O

R² is H or O

Anguidine can be targeted at the C-3, C-4, C-8 or C-15 positions, as anester or hydrazone.

THE ANTHRACYCLINES ANTIBIOTICS OF FORMULA (12):

wherein

R¹ is —CH₃, —CH₂OH, —CH₂OCO(CH₂)₃CH₃ or —CH₂OCOCH (OC₂H₅)₂

R² is —OCH₃, —OH or —H

R³ is —NH₂, —NHCOCF₃, 4-morpholinyl, 3-cyano-4-morpholinyl,1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine,cyanomethylamine, 1-cyano-2-methoxyethyl amine, or NH—(CH₂)₄—CH(OAc)₂;

R⁴ is —OH, —OTHP, or —H; and

R⁵ is —OH or —H provided that R⁵ is not —OH when R⁴ is —OH or —OTHP.

One skilled in the art understands that structural Formula (12) includescompounds which are drugs, or are derivatives of drugs, which haveacquired in the art different generic or trivial names. Table I, whichfollows, represents a number of anthracycline drugs and their generic ortrivial names and which are especially preferred for use in the presentinvention.

Of the compounds shown in Table I, the most highly preferred drug isDoxorubicin. Doxorubicin (also referred to herein as “DOX”) is thatanthracycline of Formula (1) in which R₁ is —CH₂OH, R₃ is —OCH₃, R₄ is—NH₂, R₅ —OH, and R₆ is —H.

TABLE I

Compound R¹ R³ R⁴ R⁵ R⁶ Daunorubicin^(a) CH₃ OCH₃ NH₂ OH H DoxorubicinCH₂OH OCH₃ NH₂ OH H Detorubicin CH₂OCOCH(OC₂H₅)₂ OCH₃ NH₂ OH HCarminomycin CH₃ OH NH₂ OH H Idarubicin CH₃ H NH₂ OH H Epirubicin CH₂OHOCH₃ NH₂ H OH Esorubicin CH₂OH OCH₃ NH₂ H H THP CH₂OH OCH₃ NH₂ OTHP HAD-32 CH₂OCO(CH₂)₃CH₃ OCH₃ NHCOCF₃ OH H Morpholino-Dox CH₂OH OCH₃

OH H Cyano-morpholino-Dox CH₂OH OCH₃

OH H DAPDox CH₂OH OCH₃ —NH(CH₂)₄CH(OAc)₂ OH H ^(a)“Daunomycin” is analternate name for daunorubicin

The most highly preferred drugs are the taxol, mitomycin C, andanthracycline antibiotic agents of Formula (12), described previously.

THE LIGAND

The “ligand” includes within its scope any molecule that specificallybinds or reactively associates or complexes with a receptor or otherreceptive moiety associated with a given target cell population. Thiscell reactive molecule, to which the drug reagent is linked via thelinker in the conjugate, can be any molecule that binds to, complexeswith or reacts with the cell population sought to be therapeutically orotherwise biologically modified and, which possesses a free reactivesulfhydryl (—SH) group or can be modified to contain such a sulfhydrylgroup. The cell reactive molecule acts to deliver the therapeuticallyactive drug moiety to the particular target cell population with whichthe ligand reacts. Such molecules include, but are not limited to, largemolecular weight proteins such as, for example, antibodies, smallermolecular weight proteins, polypeptide or peptide ligands, andnon-peptidyl ligands.

The non-immunoreactive protein, polypeptide, or peptide ligands whichcan be used to form the conjugates of this invention may include, butare not limited to, transferrin, epidermal growth factors (“EGF”),bombesin, gastrin, gastrin-releasing peptide, platelet-derived growthfactor, IL-2, IL-6, tumor growth factors (“TGF”), such as TGF-α andTGF-β, vaccinia growth factor (“VGF”), insulin and insulin-like growthfactors I and II. Non-peptidyl ligands may include, for example,carbohydrates, lectins, and apoprotein from low density lipoprotein.

The immunoreactive ligands comprise an antigen-recognizingimmunoglobulin (also referred to as “antibody”), or anantigen-recognizing fragment thereof. Particularly preferredimmunoglobulins are those immunoglobulins which can recognize atumor-associated antigen. As used, “immunoglobulin” may refer to anyrecognized class or subclass of immunoglobulins such as IgG, IgA, IgM,IgD, or IgE. Preferred are those immunoglobulins which fall within theIgG class of immunoglobulins. The immunoglobulin can be derived from anyspecies. Preferably, however, the immunoglobulin is of human, murine, orrabbit origin. Further, the immunoglobulin may be polyclonal ormonoclonal, preferably monoclonal.

As noted, one skilled in the art will appreciate that the invention alsoencompasses the use of antigen recognizing immunoglobulin fragments.Such immunoglobulin fragments may include, for example, the Fab′,F(ab′)₂, F_(v) or Fab fragments, or other antigen recognizingimmunoglobulin fragments. Such immunoglobulin fragments can be prepared,for example, by proteolytic enzyme digestion, for example, by pepsin orpapain digestion, reductive alkylation, or recombinant techniques. Thematerials and methods for preparing such immunoglobulin fragments arewell-known to those skilled in the art. See generally, Parham, J.Immunology, 131, 2895 (1983); Lamoyi et al., J. Immunological Methods,56, 235 (1983); Parham, id., 53, 133 (1982); and Matthew et al., id.,50, 239 (1982).

The immunoglobulin can be a “chimeric antibody” as that term isrecognized in the art. Also, the immunoglobulin may be a “bifunctional”or “hybrid” antibody, that is, an antibody which may have one arm havinga specificity for one antigenic site, such as a tumor associated antigenwhile the other arm recognizes a different target, for example, a haptenwhich is, or to which is bound, an agent lethal to the antigen-bearingtumor cell. Alternatively, the bifunctional antibody may be one in whicheach arm has specificity for a different epitope of a tumor associatedantigen of the cell to be therapeutically or biologically modified. Inany case, the hybrid antibodies have a dual specificity, preferably withone or more binding sites specific for the hapten of choice or one ormore binding sites specific for a target antigen, for example, anantigen associated with a tumor, an infectious organism, or otherdisease state.

Biological bifunctional antibodies are described, for example, inEuropean Patent Publication, EPA 0 105 360, to which those skilled inthe art are referred. Such hybrid or bifunctional antibodies may bederived, as noted, either biologically, by cell fusion techniques, orchemically, especially with cross-linking agents or disulfidebridge-forming reagents, and may be comprised of whole antibodies and/orfragments thereof. Methods for obtaining such hybrid antibodies aredisclosed, for example, in PCT application W083/03679, published Oct.27, 1983, and published European Application EPA 0 217 577, publishedApr. 8, 1987, both of which are incorporated herein by reference.Particularly preferred bifunctional antibodies are those biologicallyprepared from a “polydoma” or “quadroma” or which are syntheticallyprepared with cross-linking agents such as bis-(maleimido)-methyl ether(“BMME”), or with other cross-linking agents familiar to those skilledin the art.

In addition the immunoglobin may be a single chain antibody (“SCA”).These may consist of single chain Fv fragments (“scFv”) in which thevariable light (“V_(L)”) and variable heavy (“V_(H)”) domains are linkedby a peptide bridge or by disulfide bonds. Also, the immunoglobulin mayconsist of single V_(H) domains (dAbs) which possess antigen-bindingactivity. See, e.g., G. Winter and C. Milstein, Nature, 349, 295 (1991);R. Glockshuber et al., Biochemistry 29, 1362 (1990); and, E. S. Ward etal., Nature 341, 544 (1989).

Especially preferred for use in the present invention are chimericmonoclonal antibodies, preferably those chimeric antibodies havingspecificity toward a tumor associated antigen. As used herein, the term“chimeric antibody” refers to a monoclonal antibody comprising avariable region, i.e. binding region, from one source or species and atleast a portion of a constant region derived from a different source orspecies, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are especially preferred in certain applications of theinvention, particularly human therapy, because such antibodies arereadily prepared and may be less immunogenic than purely murinemonoclonal antibodies. Such murine/human chimeric antibodies are theproduct of expressed immunoglobulin genes comprising DNA segmentsencoding murine immunoglobulin variable regions and DNA segmentsencoding human immunoglobulin constant regions. Other forms of chimericantibodies encompassed by the invention are those in which the class orsubclass has been modified or changed from that of the originalantibody. Such “chimeric” antibodies are also referred to as“class-switched antibodies”. Methods for producing chimeric antibodiesinvolve conventional recombinant DNA and gene transfection techniquesnow well known in the art. See, e.g., Morrison, S. L, et al., Proc.Nat'l Acad. Sci., 81, 6851 (1984).

Encompassed by the term “chimeric antibody” is the concept of “humanizedantibody”, that is those antibodies in which the framework or“complementarity determining regions (“CDR”) have been modified tocomprise the CDR of an immunoglobulin of different specificity ascompared to that of the parent immunoglobulin. In a preferredembodiment, a murine CDR is grafted into the framework region of a humanantibody to prepare the “humanized antibody”. See, e.g., L. Riechmann etal., Nature 332, 323 (1988); M. S. Neuberger et al., Nature 314, 268(1985). Particularly preferred CDR'S correspond to those representingsequences recognizing the antigens noted above for the chimeric andbifunctional antibodies. The reader is referred to the teaching of EPA 0239 400 (published Sep. 30, 1987), incorporated herein by reference, forits teaching of CDR modified antibodies.

One skilled in the art will recognize that a bifunctional-chimericantibody can be prepared which would have the benefits of lowerimmunogenicity of the chimeric or humanized antibody, as well as theflexibility, especially for therapeutic treatment, of the bifunctionalantibodies described above. Such bifunctional-chimeric antibodies can besynthesized, for instance, by chemical synthesis using cross-linkingagents and/or recombinant methods of the type described above. In anyevent, the present invention should not be construed as limited in scopeby any particular method of production of an antibody whetherbifunctional, chimeric, bifunctional-chimeric, humanized, or anantigen-recognizing fragment or derivative thereof.

In addition, the invention encompasses within its scope immunoglobulins(as defined above) or immunoglobulin fragments to which are fused activeproteins, for example, an enzyme of the type disclosed in Neuberger, etal., PCT application, WO86/01533, published Mar. 13, 1986. Thedisclosure of such products is incorporated herein by reference.

As noted, “bifunctional”, “fused”, “chimeric” (including humanized), and“bifunctional-chimeric” (including humanized) antibody constructionsalso include, within their individual contexts constructions comprisingantigen recognizing fragments. As one skilled in the art will recognize,such fragments could be prepared by traditional enzymatic cleavage ofintact bifunctional, chimeric, humanized, or chimeric-bifunctionalantibodies. If, however, intact antibodies are not susceptible to suchcleavage, because of the nature of the construction involved, the notedconstructions can be prepared with immunoglobulin fragments used as thestarting materials; or, if recombinant techniques are used, the DNAsequences, themselves, can be tailored to encode the desired “fragment”which, when expressed, can be combined in vivo or in vitro, by chemicalor biological means, to prepare the final desired intact immunoglobulin“fragment”. It is in this context, therefore, that the term “fragment”is used.

Furthermore, as noted above, the immunoglobulin (antibody), or fragmentthereof, used in the present invention may be polyclonal or monoclonalin nature. Monoclonal antibodies are the preferred immunoglobulins,however. The preparation of such polyclonal or monoclonal antibodies nowis well known to those skilled in the art who, of course, are fullycapable of producing useful immunoglobulins which can be used in theinvention. See, e.g., G. Kohler and C. Milstein, Nature 256, 495 (1975).In addition, hybridomas and/or monoclonal antibodies which are producedby such hybridomas and which are useful in the practice of the presentinvention are publicly available from sources such as the American TypeCulture Collection (“ATCC”) 12301 Parklawn Drive, Rockville, Md. 20852or, commerically, for example, from Boehringer-Mannheim Biochemicals,P.O. Box 50816, Indianapolis, Ind. 46250.

Particularly preferred monoclonal antibodies for use in the presentinvention are those which recognize tumor associated antigens. Suchmonoclonal antibodies, are not to be so limited, however, and mayinclude, for example, the following:

Antigen Site Monoclonal Recognized Antibodies Reference Lung TumorsKS1/4 N. M. Varki, et al., Cancer Res. 44:681, 1984 534, F8; 604A9 F.Cuttitta, et al., in: G. L. Wright (ed) Monoclonal Antibodies andCancer, Marcel Dekker, Inc., NY., p. 161, 1984. Squamous Lung G1, LuCa2,Kyoizumi et al., Cancer Res., LuCa3, LuCa4 45:3274, 1985. Small CellLung TFS-2 Okabe et al., Cancer Res. Cancer 45:1930, 1985. Colon Cancer11.285.14 G. Rowland, et al., Cancer 14.95.55 Immunol.Immunother., 19:1,1985 NS-3a-22, NS-10 Z. Steplewski, et al., Cancer NS-19-9, NS-33a Res.,41:2723, 1981. NS-52a, 17-1A Carcinoembryonic MoAb 35 or Acolla, R. S.et al., Proc. ZCE025 Natl. Acad. Sci., (USA), 77:563, 1980. Melanoma9.2.27 T. F. Bumol and R. A. Reisfeld, Proc. Natl. Acad. Sci., (USA),79:1245, 1982. p97 96.5 K. E. Hellstrom, et al., MonoclonalAntibodiesand Cancer, loc. cit. p. 31. Antigen T65 T101 Boehringer-Mannheim, P.O.Box 50816, Indianapolis, IN 46250 Ferritin AntiferrinBoehringer-Mannheim, P.O. Box 50816, Indianapolis, IN 46250 R24 W. G.Dippold, et al., Proc. Natl. Acad. Sci. (USA), 77:6114, 1980Neuroblastoma P1 153/3 R. H. Kennet and F. Gilbert, Science, 203:1120,1979. MIN 1 J. T. Kemshead in Monoclonal Antibodies and Cancer, loc.cit. p. 49. UJ13A Goldman et al., Pediatrics, 105:252, 1984. Glioma BF7,GE2, CG12 N. de Tribolet, et al., in Monoclonal Antibodies and Cancer,loc. cit. p. 81 Ganglioside L6 I. Hellstrom et al. Proc. Natl Acad. Sci.(U.S.A) 83:7059 (1986); U.S. Pat. Nos. 4,906,562, issued March 6, 1990and 4,935,495, issued June 19, 1990. Chimeric L6 U.S. Ser. No.07/923,244, (abandoned) filed Oct. 27, 1986, equivalent to PCT PatentPublication, WO 88/03145, published May 5, 1988. Lewis Y BR64 U.S. Ser.Nos. 07/289,635 (abandoned) filed December 22, 1988, and U.S. Ser. No.07/443,696 (now U.S. Pat. No. 5,242,824) Nov. 29, 1989, equivalent toEuropean Patent Publication, EP A 0 375 562, published June 27, 1990,fucosylated BR96, Chimeric U.S. Ser. Nos. 07/374,947 Lewis Y BR96(abandoned) filed June 30, 1989, and U.S. Ser. No. 07/544,246(abandoned) filed June 26, 1990, equi-valent to PCT Patent Publication,WO 91/00295, published January 10, 1991. Breast Cancer B6.2, B72.3 D.Colcher, et al., in Monoclonal Antibodies and Cancer, loc. cit. p. 121.Osteogenic 791T/48, M. J. Embleton, ibid, p. 181 Sarcoma 791T/36Leukemia CALL 2 C. T. Teng, et al., Lancet, 1:01, 1982 anti-idiotype R.A. Miller, et al., N. Eng. J. Med., 306:517, 1982 Ovarian Cancer OC 125R. C. Bast, et al., J. Clin. Invest., 68:1331, 1981. Prostrate CancerD83.21, P6.2, J. J. Starling, et al., in Turp-27 Monoclonal Antibodiesand Cancer, loc. cit., p. 253 Renal Cancer A6H, D5D P. H. Lange, et al.,Surgery, 98:143, 1985.

In the most preferred embodiment, the ligand containing conjugate isderived from chimeric antibody BR96, “ChiBR96”, disclosed in U.S. Ser.No. 07/544,246, filed Jun. 26, 1990, and which is equivalent to PCTPublished Application, WO 91/00295, published Jan. 10, 1991. ChiBR96 isan internalizing murine/human chimeric antibody and is reactive, asnoted, with the fucosylated Lewis Y antigen expressed by human carcinomacells such as those derived from breast, lung, colon, and ovariancarcinomas. The hybridoma expressing chimeric BR96 and identified asChiBR96 was deposited on May 23, 1990, under the terms of the BudapestTreaty, with the American Type Culture Collection (“ATCC”), 12301Parklawn Drive, Rockville, Md. 20852. Samples of this hybridoma areavailable under the accession number ATCC HB 10460. ChiBR96 is derived,in part, from its source parent, BR96. The hybridoma expressing BR96 wasdeposited, on Feb. 21, 1989, at the ATCC, under the terms of theBudapest Treaty, and is available under the accession number HB 10036.Other hybridomas deposited with and accepted under the provisions of theBudapest Treaty by the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852 include HB8677, deposited Dec. 6, 1984,which produces L6 antibody, HB9895, deposited Nov. 16, 1988, whichproduces BR64 antibody, and HB9240, deposited Nov. 14, 1986, and HB9241,deposited Oct. 24, 1986, which produce chimeric L6 antibody. Withrespect to all of the foregoing hybridomas, all restrictions upon publicaccess to the deposits will be irrevocably removed upon the grant of apatent on this application, the deposits will be replaced if viablesamples cannot be dispensed by the depository, and the deposits will bemaintained in a public depository for a period of thirty years after thedate of deposit, or five years after the last request for a sample orfor the effective life of the patent, whichever is longer. The desiredhybridoma is cultured and the resulting antibodies are isolated from thecell culture supernatant using standard techniques now well known in theart. See, e.g., “Monoclonal Hybridoma Antibodies: Techniques andApplications”, Hurell (ed.) (CRC Press, 1982).

Thus, as used “immunoglobulin” or “antibody” encompasses within itsmeaning all of the immunoglobulin/antibody forms or constructions notedabove.

Preparation of the Conjugates

The conjugates of the present invention may be constructed by attachingthe drug moiety to the antibody through a linker made up of a peptidesequence which may be cleaved by the lysosomal proteases cathepsin B, Cand D, and a self-immolating spacer.

A process for preparing the compound of the present invention is onewherein a solution of the antibody in a phosphate buffer or PBS wastreated with a solution of dithiothreitol (DTT) at 25-45° C., for about1-10 hours under N₂. The solution was then diafiltered against phosphatebuffered saline (PBS) for ½ to 12 hours depending on the size ofdiafiltration cell and volume of solution under N₂, until the effluentis free of SH groups, then treated with the appropriate amount ofpeptide-PABC-drug [based on the number of SH groups in the Mab(determined by Ellman titration)] in distilled water, at 0±10° C. for 15minutes to 8 hours. The solution was then dialyzed against PBS for about24 hours, at room temperature, then filtered and the filtrate was shakenfor 15 minutes to 8 hours at room temperature with Biobeads, followed byanother filtration.

Schemes 1-11 show the synthesis of model compounds that were tested withcathepsin B in order to determine the optimal characteristics of thelinker including the peptide sequence, self-immolating spacer, andattachment to antibody.

Scheme 12 shows the synthesis of the linker compound MC-Phe-Lys-PABC-DOX(50) which is conjugated to the antibody carrier. The NHS active esterof Fmoc-Phe (43) was coupled to N^(ε)-Mtr-Lys (42) in an organic/aqueoussolvent mixture to give the dipeptide Fmoc-Phe-N^(ε)-Mtr-Lys (44). Thisin turn was coupled to p-aminobenzyl alcohol using EEDQ resulting inalcohol 45. The Fmoc group was removed with diethylamine, and the freeN-terminal Phe was coupled to MC-NHS to give maleimidopeptide alcohol47. Addition of bis-p-nitrophenyl carbonate provided the activatedcarbonate 48 and the p-nitrophenyl group was displaced by DOX in NMP atroom temperature. The resulting substrate MC-Phe-N^(ε)-Mtr-Lys-PABC-DOX(49) was deprotected in quantitative yield by treatment withdichloroacetic acid/anisole in CH₂Cl₂ for 1 hour to give 50.

Scheme 13 shows the synthesis of a MMC-containing linker compoundMC-Phe-Lys-PABC-MMC (52) from activated carbonate 48. The aziridinenitrogen of MMC is not nucleophilic enough to directly displace thep-nitrophenol of 48 but, in the presence of a 10-fold excess of HOBt,some of the corresponding HOBt active ester forms, and is active enoughto react with MMC. Chloroacetic acid is used instead of dichloroaceticacid for the deprotection of 51 because of acid sensitivity of MMC.

Scheme 14 shows the preparation of a taxol containing linker compoundMC-Phe-Lys-PABC-7-taxol (55). Maleimidopeptide alcohol 47 was treatedwith 2′-Mtr-taxol-7-chloroformate (prepared from 53) to giveMC-Phe-N^(ε)-Mtr-Lys-PABC-7-Taxol (54). This was deprotected withchloroacetic acid to give 55.

Scheme 15 shows the synthesis of a citrulline containing linker compoundMC-Val-Cit-PABC-DOX (62) which is carried out essentially as describedabove for 49 and requires no side chain deprotection.

Scheme 16 shows the preparation of a linker compound containing an addedaminocaproyl spacer designed so as to move the site of enzymaticcleavage away from the bulky antibody. MC-NH-C-Phe-Lys-PABC-DOX (72) wasprepared using procedures essentially identical to those used in thesynthesis of 50 and 55.

Scheme 17 shows the synthesis of a MMC-containing linker compoundMC-Phe-Lys-GABA-MMC (78) which incorporates a GABA spacer in place ofPABC. This was prepared essentially as described for 52 above.

Scheme 18 shows the synthesis of a potential protease-active prodrug ofcortisone, Z-Phe-Lys-Cortisone (81). This was prepared essentially asdescribed for MC-Phe-Lys-PABC-DOX (50).

Scheme 19 shows the synthesis of a linker compound containingtaxol-2′-ethyl carbonate, an active prodrug of taxol. Compounds of thepresent invention includeBR96-succinimidocaproyl-phenylalanine-lysine-p-aminovenzyl-carbamoyloxy-doxorubicin,BR96-succinimidocaproyl-valine-lysine-p-aminobenzyl-carbamoyloxy-doxorubicin,BR96-succinimidocaproyl-valine-citrulline-p-aminobenzyl-carbamoyloxy-doxorubicin,BR96-succinimidocaproyl-phenylalanine-lysine-p-aminobenzyl-carbamoyloxy-2′-taxol,BR96-succinimidocaproyl-phenylalanine-lysine-p-amonobenzyl-carbamoyolxy-7-taxol,andBR96-succinimidocaproyl-phenylalanine-lysine-p-aminobenzyl-carbamoyolxy-motomycin-c.

BIOLOGICAL ACTIVITY

Representative conjugates of the present invention were tested in bothin vitro and in vivo systems to determine biological activity. In thesetests, the potency of conjugates of cytotoxic drugs was determined bymeasuring the cytotoxicity of the conjugates against cells of humancancer origin. The following describes representative tests used and theresults obtained. One skilled in the art will recognize that any tumorline expressing the desired antigen could be used in substitution of thespecific tumor lines used in the following analyses.

TEST I Cathepsin B Release of Free DOX

300 μl of the above conjugate solution was diluted to 1 ml with pH 5.0acetate buffer (25 mM+1 mM EDTA) giving a final pH of 5.3. This solutionwas incubated at about 37° C. while 6 μl of cathepsin B solution (see 2below) was incubated with 20 μl of activating solution (see 2 below) forabout 15 minutes at room temperature. The enzyme solution was thentreated with the pH 5.3 conjugate solution and the mixture incubated atabout 37° C. 25 μl aliquots were removed periodically and diluted with50 μl of cold methanol to precipitate the protein. The samples werecentrifuged and the liquid injected into the HPLC (C-18 column; 80:20methanol/pH 2.8 triethylammonium formate buffer; 1 ml/min.; 495 mndetection wavelength). Peak areas were calibrated by injection of knownconcentration of DOX. The half-life of release of free DOX wasdetermined to be about 3 hours with 93% of the theoretical release ofDOX accounted for (some free DOX is likely to precipitate out with theprotein).

TEST II Human Plasma Stability

300 μl of conjugate solution was diluted to 1 ml with freshly drawnhuman plasma and the mixture was incubated at about 37° C. 25 μlaliquots were removed periodically and diluted with 50 μl of coldmethanol. The samples were centrifuged and the liquid injected into theHPLC (conditions as above). Separate plasma samples were incubated with1% and 2% theoretical release of free DOX for several minutes andtreated in the same way. Free DOX was successfully detected andquantified at these levels. No free DOX was detected from the conjugatein plasma over 7.5 hours. (half-life>375 hrs.).

TEST III Cathepsin B Unmasking of Z-Phe-Lys-PABC-DOX

Bovin spleen cathepsin B (Sigma, EC 3.4.22.1, MW ca. 40,000) (10 units)was dissolved in 1 ml pH 5.0 acetate buffer (25 mM acetate+1 mM EDTA),giving a solution roughly 13.7 M. 6 μl of the enzyme solution wasincubated with 12 μl of an activating solution (30 mM dithiothreitol and15 mM EDTA) for about 15 minutes at room temperature. To this was added2 ml of pH 5.0 acetate buffer (25 mM acetate with 1 mM EDTA) which hadbeen incubated at about 37° C., followed by 8 μl of a 10 mM solution ofZ-Phe-Lys-PABC-DOX in methanol ([Substrate]=40 μM, [Cathepsin B]=ca. 41nM). The mixture was incubated at about 37° C., and aliquots wereperiodically removed and injected into the HPLC (C-18 column; 80:20methanol/pH 2.8 triethylammonium formate (50 mM) buffer; 1 ml/min.; 495mn detection wavelength). The half-life of release of free DOX wasdetermined to be 7-9 minutes.

TEST IV Human plasma stability

4 μl of a 10 mM solution of Z-Phe-Lys-PABC-DOX was dissolved in 1 ml offreshly drawn human plasma. Aliquots (50 μl) were periodically removedand diluted with cold methanol (100 μl). The samples were centrifugedand the resulting liquid injected into the HPLC (conditions as above).Enough DOX was added to a separate sample of plasma to give atheoretical release of 1% from the substrate. This was successfullydetected using the same methods. No free DOX was detected fromZ-Phe-Lys-PABC-DOX in plasma over 7 hours (half-life>350 hrs.)

TEST V Materials and Methods

Human Tumor Cell Lines.

L2987 is a lung adenocarcinoma line obtained from I. Hellstrom(Bristol-Myers Squibb, Seattle, Wash.). The HCT116 colorectal tumor linewas obtained from M. Brattain (Baylor Inst., Tex.). A2780 is an ovariancarcinoma line obtained from K. Scanlon (National Cancer Institute).

Binding Assays.

Binding assays were performed by indirect immunofluorescence. Briefly,target cells were harvested in logarithmic phase using trypsin/EDTA(GIBCO, Grand Island, N.Y.) in PBS. The cells were washed twice in PBScontaining 1% bovine serum albumin (BSA, Sigma Chemical Co., St. Louis,Mo.) and resuspended to 1×10⁷/ml in PBS containing 1% BSA and 0.1% NaN₃Cells (0.1 ml) were mixed with various antibodies (0.1 ml at 40 ugMAb/ml) and incubated for about 45 minutes at about 4° C. The cell werewashed 2× in and resuspended in 0.1 ml of an appropriate concentrationof rabbit anti-human IgG (Cappel Laboratories, Cochranville, Pa., Fab′2fragment). Cells were incubated for about 30 minutes at about 4° C.,washed 2× and kept on ice until analyzed on a Coulter EPICS 753fluorescence-activated cell sorter. Data are expressed as fluorescenceintensity (FI): the mean channel number of specific minus controlantibody.

In vitro cytotoxicity assays.

Monolayer cultures of human carcinoma cells were harvested usingtrypsin-EDTA (GIBCO, Grand Island, N.Y.), and the cells counted andresuspended to 1×10⁵/ml in RPMI-1640 containing 10% heat inactivatedfetal calf serum (RPMI-10% FCS). Cells (0.1 ml/well) were added to eachwell of 96 well microtiter plates and incubated overnight at about 37°C. in a humidified atmosphere of 5% CO₂. Media was removed from theplates and serial dilutions of DOX or MAb-DOX conjugates added to thewalls. All dilutions were performed in quadruplicate. Cells were exposedto DOX or MAb-DOX conjugates for about 2 hours at about 37° C. in ahumidified atmosphere of 5% CO₂. Plates were then centrifuged (200×g, 5min.), the drug or conjugate removed, and the cells washed 3× withRPMI-10% FCS. The cells were cultured in RPMI-10% FCS (37° C., 5% CO₂)for an additional 48 hours. At this time the cells were pulsed for about2 hours with 1.0 uCi/well of ³H-thymidine (New England Nuclear, Boston,Mass.). The cells were harvested onto glass fibre mats (SkatronInstruments, Inc., Sterling, Va.), dried, and filter bound ³Hradioactivity determined (β-Plate scintillation counter, Pharmacia LKBBiotechnology, Piscataway, N.J.). Inhibition of ³H-thymidine uptake wasdetermined by comparing the mean CPM for treated samples with that ofthe mean CPM of the untreated control.

Results

Binding Assays:

The L2987, A2780 and HCT116 human carcinoma lines were evaluated for theexpression of the BR96 antigen using direct immunofluorescence. As shownin FIG. 1, the L2987 lung line (A) expressed the greatest density of theBR96 antigen (FI=172.8), the A2780 ovarian line (B) expressed BR96 at alower density (FI=103.2), and the HCT116 colon line (C) did not expresssignificant amounts of the BR96 antigen (FI=0).

Cytoxicity of BR96-DOX peptide linked conjugate:

The in vitro potency of the BR96-DOX peptide immunoconjugate wasevaluated in parallel against the L2987, A2780, and HCT116 humancarcinoma lines. As described above these cells express variousdensities of the BR96 antigen (L2987>A2780>>HCT116). Unconjugateddoxorubicin was also evaluated. As shown in FIG. 2, the potency of theBR96-DOX conjugate was equivalent to that of unconjugated DOX againstthe L2987 lung line (A). The BR96-DOX conjugate was approximately 50fold less potent than unconjugated DOX against the A2780 ovarian line(B). The BR96-DOX conjugate was not active against the antigen-negativeHCT116 line (C). However, as shown this line was sensitive tounconjugated DOX. These data demonstrate the direct relationship betweenthe in vitro potency of the BR96-DOX conjugate and the epitope densityof the BR96 antigen. In summary the BR96-DOX conjugate demonstratesantigen-specific cytotoxicity in vitro and the potency of the conjugateis related to the density of BR96 antigen expressed by various celllines.

TEST VI

The BR96-PEP-DOX conjugate (MR=4.41) was evaluated in vivo (Table 1)against L2987 human lung carcinoma xenografts. Therapy was initiated 14days after tumor implant when the tumors were approximately 75 mm 3 insize.

The BR96-PEP-DOX conjugate was active and tolerated at doses of 1.25-20mg/kg equivalent DOX/injection. Higher doses were not evaluated in thisfirst experiment. As shown in Table 1 the BR96-PEP-DOX conjugate wassignificantly more active than optimized DOX at doses of ≧2.5 mg/kgequivalent DOX/injection. The activity of the BR96-PEP-DOX conjugateadministered at 1.25 mg/kg was similar to that of unconjugated DOXadministered at 8 mg/kg. These data suggest that the in vivo potency ofthe BR96-PEP-DOX conjugates is similar to that of BMS-182248. Thepeptide-DOX conjugates will be evaluated for antigen-specific antitumoractivity as soon as a non-binding (IgG-PEP-DOX) conjugate can beprepared.

TABLE 1 Antitumor activity of BR96-DOX peptide conjugates againstestablished L2987 human tumor xenografts % Tumor Regressions Dose/ LogNumber Treatment Injection Cell of DOX (mg/kg) BR96 Kill CompletePartial Mice DOX 8 — 2.4 10 0 10 6 — 1.5 0 0 10 10 BR96-DOX 20 1250  >7100 0 9 10 625  >7 89 11 10 5 312  >7 100 0 10 2.5 156  >7 90 10 10 1.2578 2.4 10 10 10 0.63 39 0.3 0 0 10 0.31 20 0.2 0 0 10

As a result of the above tests it can be seen that the compounds of thepresent invention are highly effective antitumor agents. They kill tumorcells in vitro via a specific targeting mechanism, in which the attachedMAb BR96 is the targeting moiety, as shown by the fact that cells whichexpress high levels of the antigen recognized by the MAb are efficientlykilled; cells with less antigen are less efficiently killed; and cellswithout the antigen are not killed. Since all three cell types aresensitive to DOX, these results must arise from release of DOX afterdifferential binding to the cells, not from differential toxicity of DOXto the various cell lines. The mechanism of the present invention issupported by the finding that Cathepsin B, a lysosomal protease,releases free DOX rapidly from both the peptide linker and the completeimmunoconjugate. Since adventitious proteases in human blood do notrelease DOX from either the peptide linker or the completeimmunoconjugate, it can be inferred that the immunoconjugate will reachtumor cells in human intact, without releasing free DOX enroute.Finally, in vivo experiments in tumor-bearing mice show that theimmunoconjugate of the present invention produces remissions ofantigen-positive tumors, with greater potency and less toxicity to thehost than free DOX.

Thus, in an embodiment of the present invention, there is provided amethod for the treatment of a neoplastic disease which comprisesadministering to a warm-blooded animal in need thereof, atherapeutically effective or biological function modifying amount of aconjugate of Formula (I). As can be appreciated, the particularconjugate used will depend on the disease state to be treated or thebiological system to be modified. In particular, one skilled in the artwill be able to select a particular ligand and drug to prepare aconjugate of Formula (I) which has specificity for the treatment of thedisease or is able to modify the biological function desired.

A particularly preferred conjugate for this purpose is animmunoconjugate in which the drug moiety is doxorubicin and the ligandportion is selected from the group consisting of BR96, chimeric BR96,and the antigen-recognizing fragments thereof. The most preferred ligandfor this embodiment is chimeric BR96, and the antigen-recognizingfragments thereof.

In a further embodiment, there is provided a process for preparing acompound of Formula (I), as previously defined.

The conjugates of the invention are administered to the patient in theform of a pharmaceutical formulation which comprises a conjugate ofFormula (I) and a pharmaceutically acceptable carrier, excipient ordiluent therefor. As used, “pharmaceutically acceptable” refers to thoseagents which are useful in the treatment or diagnosis of a warm-bloodedanimal including, for example, a human, equine, porcine, bovine, murine,canine, feline, or other mammal, as well as an avian or otherwarm-blooded animal. The preferred mode of administration isparenterally, particularly by the intravenous, intramuscular,subcutaneous, intraperitoneal, or intralymphatic route. Suchformulations can be prepared using carriers, diluents or excipientsfamiliar to one skilled in the art. In this regard, See, e.g.Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack PublishingCompany, edited by Osol et al. Such compositions may include proteins,such as serum proteins, for example, human serum albumin, buffers orbuffering substances such as phosphates, other salts, or electrolytes,and the like. Suitable diluents may include, for example, sterile water,isotonic saline, dilute aqueous dextrose, a polyhydric alcohol ormixtures of such alcohols, for example, glycerin, propylene glycol,polyethylene glycol and the like. The formulations may containpreservatives such as phenethyl alcohol, methyl and propyl parabens,thimerosal, and the like. If desired, the formulation can include 0.05to about 0.20 percent by weight of an antioxidant such as sodiummetabisulfite or sodium bisulfite.

For intravenous administration, the formulation preferably will beprepared so that the amount administered to the patient will be fromabout 1 to about 250 g of the desired conjugate. Preferably, the amountadministered will be in the range of about 4 g to about 25 g of theconjugate. The conjugates of the invention are effective over a widedosage range depending on factors such as the disease state to betreated or the biological effect to be modified, the manner in which theconjugate is administered, the age, weight and condition of the patientas well as other factors to be determined by the treating physician.Thus, the amount administered to any given patient must be determined onan individual basis.

All publications cited in this specification are indicative of the levelof skill of those in the art to which this application pertains. Eachpublication is individually incorporated herein by reference in thelocation where it is cited.

One skilled in the art will appreciate that although specific reagentsand reaction conditions are outlined in the following Preparations andExamples, modifications can be made which are meant to be encompassed bythe spirit and scope of the invention. The following Preparations andExamples, therefore, are provided to further illustrate the invention.

EXAMPLE 1 Preparation of Allyl-p-Nitrophenyl Carbonate (1)

Allyl alcohol (0.5 ml, 7.35 mmoles) in CH₂Cl₂ (3 ml) at room temperaturewas treated with p-nitrophenyl chloroformate (1.482 g, 1 equiv.). Tothis was added pyridine (0.6 ml, 1 equiv.) in CH₂Cl₂ (2 ml), dropwiseover 10 minutes. After about 5 hours at room temperature the mixture waswashed with 15% citric acid, water and brine, dried, and evaporated togive a thick, pale yellow oil. This was chromatographed on silica,eluting with 10-50% EtOAc/hexane, to give the product as an off-white,crystalline solid (1.542 g, 94%). ¹H-NMR (CDCl₃): δ 4.78 (2H, d, CH₂—O),5.40 (2H, q, vinyl CH₂), 5.99 (1H, m, vinyl CH), 7.37 and 8.26 (4H, 2×d,Ph); MS (DCI): 224 (MH)⁺;

Anal. calc. for C₁₀H₉NO₅: C-53.82, H-4.06, N-6.28; Found: C-53.73,H-4.03, N-6.23.

EXAMPLE 2 Preparation of N^(α)-Boc-N^(ε)-Alloc-Lys (2)

A solution of Boc-Lys (8.4414 g, 34.27 mmoles) and NaHCO₃ (2.88 g, 1equiv.) in water (50 ml) was added to allyl-p-nitrophenyl carbonate (1)(7.649 g, 1 equiv.) in DME (50 ml) at room temperature The mixture wasstirred overnight at room temperature Water (80 ml) was then added andthe mixture was extracted with ether (3×50 ml). The aqueous layer wasacidified to pH 2 with 10% citric acid and then extracted with EtOAc(3×80 ml). The combined organic components were washed with water andbrine, dried, and evaporated to give a white solid. This was treatedwith ether (100 ml) and the resulting mixture was sonicated for about 15minutes to dissolve p-nitrophenol and then the solid (10.303 g, 91%) wascollected by filtration and washed repeatedly with ether. ¹H-NMR(CDCl₃/CD₃OD): δ 1.41 (9H, s, t-Bu), 1.49 and 1.70 (6H, m, Lys CH₂),3.13 (2H, m, Lys N—CH₂), 4.25 (1H, m, CH), 4.52 (2H, d, allyl O—CH₂),5.24 (2H, q, vinyl CH₂), 5.87 (1H, m, vinyl CH); MS (DCI): 331 (MH⁺),275 (MH⁺—C₄H₈).

EXAMPLE 3 Preparation of N^(ε)-Alloc-Lys-TFA (3)

N^(α)-Boc-N^(ε)-alloc-Lys 2 (9.94 g, 30 mmoles) in CH₂Cl₂ (50 ml) wastreated with TFA (19 ml) at room temperature The mixture was sonicatedbriefly and then stirred for about 1 hour. The solvents were evaporatedat about 40° C. and the resulting yellow gum was triturated with ether(75 ml), giving a white solid (8.58 g, 83%. ¹H-NMR (D₂O): δ 1.46 and1.87 (4H and 2H resp., m, Lys CH₂), 3.11 (2H, m, N—CH₂), 3.80 (1H, t,Lys CH), 4.51 (2H, br s, allyl O—CH₂), 5.22 (2H, q, vinyl CH₂), 5.90(1H, m, vinyl CH); MS (DCI): 231 (MH)⁺;

Anal. calc. for C₁₂H₁₉N₂O₆F₃: C-41.86, H-5.56, N-8.14; Found: C-42.30,H-5.52, N-8.29.

EXAMPLE 4 Preparation of Z-Phe-NHS (4)

Z-Phe (11.03 g, 36.85 mmoles), and NHS (4.45 g, 1.1 equiv.) in THF (50ml) at about 0° C. were treated with DCC (7.98 g, 1.05 equiv.). After afew minutes a heavy white precipitate appeared. The mixture was allowedto warm to room temperature and was stirred for about 16 hours. Thesolid DCU by-product was filtered off and the filtrate was evaporated.The resulting thick, colorless oil was dissolved in CH₂Cl₂ (80 ml). Themixture was allowed to stand for an hour and was then filtered to removemore DCU. The filtrate was evaporated and the resulting colorless glasswas dried in vacuo for about 3 hours, giving a foamy solid (14.023 g,96%) that was used without further purification. ¹H-NMR (CDCl₃/CD₃OD):δ2.88 (4H, s, NHS CH₂), 3.27 (2H, m, Phe CH₂), 4.70 (1H, m, Phe CH),5.13 (2H, s, Z CH₂), 7.27 (10H, m, Ph).

EXAMPLE 5 Preparation of Z-Phe-N^(ε)-Alloc-Lys (5)

Z-Phe-NHS (4) (2.783 g, 7.021 mmoles) in DME (30 ml) at room temperaturewas treated with a solution of N^(ε)-alloc-Lys-TFA (2.54 g, 1.05 equiv.)and NaHCO₃ (1.24 g, 2.1 equiv.) in water (30 ml). The mixture wasstirred vigorously at room temperature for 2 days. A small amount of DCUwas removed by filtration and the filtrate was diluted with water (50ml) and then acidified to pH 3 with 15% citric acid. The resultingmixture was extracted with EtOAc (3×80 ml) and the combined organiclayers were washed with water and brine, dried, and evaporated to give aglassy solid. This was treated with ether (150 ml), sonicated, andheated in a water bath (50° C.). Upon cooling, the white solid product(2.79 g, 78%) was collected by filtration and washed with ether. ¹H-NMR(CDCl₃/CD₃OD): δ 1.25, 1.43, 1.74 and 1.81 (6H, m, Lys CH₂), 3.00 (2H,m, Phe CH₂), 3.08 (2H, m, N—CH₂), 4.43 (2H, m, CO—CH), 4.48 (2H, d,allylic O—CH₂), 5.02 (2H, m, Z CH₂), 5.20 (2H, q, vinyl CH₂), 5.84 (1H,m, vinyl CH), 7.22 (1OH, m, Ph); MS (FAB): 512 (MH)⁺, 534 (M+Na)⁺, 556(M+K)⁺;

Anal. calc. for C₂₇H₃₃N₃O₇: C-63.39, H-6.50, N-8.21; Found: C-62.98,H-6.48, N-8.21.

EXAMPLE 6 Preparation of Z-Phe-N^(ε)-Alloc-Lys-PAB-OH (6)

Z-Phe-N^(ε)-alloc-Lys (5) (524.7 mg, 1.026 mmoles) and p-aminobenzylalcohol (133 mg, 1.05 equiv.) in THF (10 ml) at room temperature weretreated with EEDQ (266.3 mg, 1.05 equiv.). The mixture was stirred atroom temperature for about 16 hours. The mixture was evaporated todryness at about 30° C. and the residue triturated with ether (15 ml).The resulting white solid product (591.6 mg, 94%) was collected byfiltration and washed with ether. ¹H-NMR (CDCl₃/CD₃OD): δ 1.25, 1.42,1.59 and 1.77 (6H, m, Lys CH₂), 2.97 (2H, m, Phe CH₂), 3.06 (2H, m,N—CH₂), 4.37 (2H, m, Phe and Lys CH), 4.46 (2H, d, allyl O—CH₂), 4.55(2H, s, Ph—CH ₂—OH), 4.98 (2H, m, Z CH₂), 5.18 (2H, q, vinyl CH₂), 5.81(1H, m, vinyl CH) 7.08 and 7.43 (4H, 2×d, PAB Ph), 7.11 and 7.23 (10H,m, Z and Phe Ph); MS (FAB): 617 (MH)⁺, 639 (M+Na)⁺, 655 (M+K)⁺;

Anal. Calc. for C₃₄H₄₀N₄O₇: C-66.22, H-6.54, N-9.08, Found: C-65.72,H-6.43, N-8.92.

EXAMPLE 7 Preparation of Z-Phe-N^(ε)-Alloc-Lys-PABC-PNP (7)

Z-Phe-N^(ε)-alloc-Lys-PAB-OH (6) (269.6 mg, 437.2 μmoles) in dry THF (8ml) at room temperature was treated with p-nitrophenyl chloroformate(106 mg, 1.2 equiv.) and pyridine (42.5 μl, 1.2 equiv.). After about 6hours TLC (silica; 25:1 CH₂Cl₂/CH₃OH) indicated completion. EtOAc (25ml) and 10% citric acid (25 ml) were added. The organic layer was washedwith water and brine, dried, and evaporated to give a yellow solid whichwas chromatographed on silica, eluting with 30:1 CH₂Cl₂/CH₃OH, to givethe product as an off-white solid (297.4 mg, 87%). ¹H-NMR (CDCl₃/CD₃OD):δ 1.24, 1.42, 1.59 and 1.78 (6H, m, Lys CH₂), 2.97 (2H, m, N—CH₂), 3.04(2H, m, Phe CH₂), 4.38 (2H, m, Phe and Lys CH), 4.46 (2H, d, allylO—CH₂), 5.01 (2H, s, Z CH₂), 5.17 (2H, q, vinyl CH₂), 5.21 (2H, s, PABCH₂—O), 5.37 and 5.80 (each 1H, m, Phe and Lys NH), 5.83 (1H, m, vinylCH), 7.11 and 7.56 (4H, 2×d, PAB Ph), 7.13 and 7.25 (10H, m, Phe and ZPh), 7.35 and 8.10 (each 2H, d, PNP Ph), 9.23 (1H, br s, PAB NH); MS(FAB): 782 (MH⁺), 804 (M+Na)⁺, 820 (M+K)⁺;

Anal. calc. for C₄₁H₄₃N₅O₁₁: C-62.99, H-5.54, N-8.96; Found: C-62.75,H-5.49, N-8.86.

EXAMPLE 8 Preparation of Z-Phe-N^(ε)-Alloc-Lys-PABC-DOX (8)

Z-Phe-N^(ε)-alloc-Lys-PABC-PNP (7) (337.2 mg, 431.3 μmoles) and DOX-HCl(275.2 mg, 1.1 equiv.) in NMP (8 ml) at room temperature were treatedwith triethylamine (66 μl, 1.1 equiv.). The mixture was allowed to standin the dark for about 2 days. The mixture was then diluted with 10%i-Pr—OH/EtOAc (100 ml) and washed with water (3×100 ml) and brine,dried, and evaporated to give an orange solid. This was chromatographedon silica, eluting with 1) 25:1 and 2) 15:1 CH₂Cl₂/CH₃OH, to give theproduct as an orange solid (496.3 mg, 97%). ¹H-NMR (CDCl₃/CD₃OD): δ 1.18(3H, d, sugar CH₃), 1.22, 1.38, 1.56 and 1.77 (6H, m, Lys CH₂), 1.74(2H, m, D-ring—CH₂), 2.23 (2H, m, D-ring CH₂), 2.95 (2H, m, sugar CH₂)),3.02 (2H, m, N—CH₂), 3.53 (1H, s, sugar HO—CH), 3.80 (1H, m, sugarHN—CH), 3.99 (3H, s, OCH₃), 4.06 (1H, m, sugar CH₃—CH), 4.39 (2H, m, Pheand Lys CH), 4.43 (2H, d, allyl O—CH₂), 4.70 (2H, s, PAB CH₂—O), 4.89(2H, m, Z CH ₂), 4.92 (1H, m, anomeric CH), 4.96 (2H, d, CO—CH ₂—OH),5.15 (2H, q, vinyl CH₂), 5.11, 5.39 (each 1H, s, OH), 5.41 (1H, br, DOXPh—CH), 5.60 and 5.92 (each 1H, m, amide NH), 5.79 (1H, m, vinyl CH),7.08 and 7.23 (10H, m, Phe and Z Ph), 7.13 and 7.40 (4H, 2×d, PAB Ph),7.50, 7.68 and 7.90 (each 1H, m, DOX Ph), 9.15 (1H, br s, PAB NH); MS(FAB): 1209 (M+Na)⁺, 1224 (M+K)⁺; HRMS (FAB): Accurate mass calc. forC₆₂H₆₇N₅O₁₉: 1186.4509; found: 1186.4463.

EXAMPLE 9 Preparation of Z-Phe-Lys-PABC-DOX-HCl (9)

Z-Phe-N^(ε)-alloc-Lys-PABC-DOX (8) (34.9 mg, 29.4 μmoles) and(PPh₃)₂PdCl₂ (0.6 mg, 3%) in dry THF (1 ml) under argon at roomtemperature were treated with acetic acid (3.5 μl, 2 equiv.) and thenwith Bu₃SnH (10 μl, 1.2 equiv.). The reaction was stirred at roomtemperature for about 1.5 hours and then treated with 1M HCl in ether(60 μl, 2 equiv.). The mixture was stored in the freezer for about 1hour and then the crude orange solid was collected by filtration andwashed repeatedly with ether. The solid was washed through the glassfrit with 5:1 CH₂Cl₂/CH₃OH and then the filtrate was evaporated. Theresidue was sonicated in methanol (5 ml) to dissolve as much as possibleand then filtered to remove an insoluble red by-product. The filtratewas evaporated to give an orange-red solid (25.1 mg, 75%). ¹H-NMR(CDCl₃/CD₃OD): δ 1.18 (2H, d, sugar CH₃), 1.34, 1.65 and 1.73 (6H, m,Lys CH₂), 2.14 (2H, m, sugar CH₂), 2.81 (2H, m, CH ₂—NH₃₊), 3.76 (1H, m,sugar HO—CH), 3.98 (3H, s, OCH₃), 4.05 (1H, m, HN—CH), 4.38 and 4.45(each 1H, m, Phe and Lys CH), 4.67 (2H, s, CO—CH ₂—OH), 4.85 (1H, m,anomeric CH), 7.04 and 7.20 (10H, m, Z and Phe Ph), 7.14 and 7.43 (4H,m, PAB Ph), 7.30, 7.69 and 7.92 (each 1H, m, DOX Ph); HPLC (C-18, 15 cmcolumn, 8:2 MeOH/50 mM Et₃N—HCO₂H buffer (pH 2.8), 1 ml/min., 495 nm):single peak, retention time 7.1-7.2 min.; MS (FAB): 1102 (MH⁺), 1124(M+Na)⁺; HRMS (FAB): Accurate mass calc. for C₅₈H₆₄N₅O₁₇: 1102.4297,found: 1102.4260.

EXAMPLE 10 Preparation of Z-Val-NHS (10)

Z-Val (699.4 mg, 2.78 mmoles) and NHS (352.4 mg, 1.1 equiv.) in THF (20ml) at about 0° C. were treated with DCC (632 mg, 1.1 equiv.). Thereaction was worked up as described above for Z-Phe-NHS (4) to give theproduct as a glassy solid which was carried on to the next step withoutpurification. ¹H-NMR δ 1.03 (6H, 2×d, Val CH₃), 2.31 (1H, m, ValCH₃—CH), 2.82 (4H, s, NHS CH₂), 4.65 (1H, AB Q, Val CO—CH), 5.12 (2H, s,Z CH₂), 5.30 (1H, d, NH), 7.34 (5H, m, Ph).

EXAMPLE 11 Preparation of Z-Val-N^(ε)-Alloc-Lys (11)

Z-Val—NHS (10) (about 2.78 mmoles) in DME (30 ml) was added to asolution of N^(ε)-alloc-Lys-TFA (3) (958.3 mg, 1 equiv.) and NaHCO₃ (468mg, 2 equiv.) in water (20 ml). The reaction was worked up as describedabove for Z-Phe-N^(ε)-alloc-Lys (5) to give the product as a white solid(1.2855g, quant.). ¹H-NMR (CDCl₃/CD₃OD): δ0.89 (6H, 2×d, Val CH₃), 1.30,1.42, 1.62 and 1.81 (6H, m, Lys CH₂), 2.03 (1H, m, Val CH₃—CH), 3.07(2H, m, Lys N—CH₂), 3.92 (1H, AB q, Lys CH), 4.42 (1H, m, Val CO—CH),4.49 (2H, d, allyl O—CH₂), 5.06 (2H, s, Z CH₂), 5.19 (2H, q, vinyl CH₂),5.82 (1H, m, vinyl CH), 7.28 (5H, m, Ph); MS (FAB): 949 (MH⁺), 971(M+Na)⁺, 987 (M+K)⁺;

Anal. Calc. for C₂₃H₃₃N₃O₇: C-59.60, H-7.18, N-9.07; Found: C-59.94,H-7.31, N-8.90.

EXAMPLE 12 Preparation of Z-Val-N^(ε)-Alloc-Lys-PAB-OH (12)

Z-Val-Ne-alloc-Lys (11) (587.9 mg, 1.27 mmoles) and p-aminobenzylalcohol (172 mg, 1.1 equiv.) in THF (20 ml) at room temperature weretreated with EEDQ (345 mg, 1.1 equiv.). The mixture was stirred at roomtemperature for 16 hrs. Workup as described above forZ-Phe-N^(ε)-alloc-Lys-PAB-OH (6) gave the product as a white solid(591.0 mg, 82%). ¹H-NMR (CDCl₃/CD₃OD): δ 0.86 (6H, m, Val CH₃),1.24-1.67 (6H, m, Lys CH₂), 2.03 (m, 1H, Val CH₃—CH), 3.08 (2H, m, LysN—CH₂), 4.00 (1H, m, Lys CH), 4.47 (3H, m, Val CO—CH and allyl O—CH₂),4.57 (2H, s, PAB—CH ₂—OH), 5.05 (2H, s, Z CH₂), 5.19 (2H, q vinyl CH₂),5.81 (1H, m, vinyl CH), 7.26 and 7.43 (4H, m, PAB Ph), 7.30 (5H, s, ZPh); MS (FAB): 569 (MH)⁺, 591 (M+Na)⁺, 607 (M+K)⁺;

Anal. Calc. for C₃₀H₄₀N₄O₇-½ H₂O: C-62.38, H-7.15, N-9.70; Found:C-62.40, H-7.22, N-9.79.

EXAMPLE 13 Preparation of Z-Val-N^(ε)-Alloc-Lys-PABC-PNP (13)

Z-Val-Ne-alloc-Lys-PAB-OH (12) (297.4 mg, 523 μmoles) and p-nitrophenylchloroformate (264 mg, 2.5 equiv.) in CH2Cl2 (15 ml) at room temperaturewere treated with pyridine (106 μl, 2.5 equiv.). The mixture was stirredat room temperature for about 16 hours. Workup as described above forZ-Phe-Ne-alloc-Lys-PABC-PNP (7) gave the product as a white solid (271.0mg, 71%). ¹H-NMR (CDCl₃/CD₃OD): δ0.91 (6H, m, Val CH₃), 1.33-1.87 (6H,Lys CH₂), 2.02 (1H, m, Val CH₃—CH), 3.08 (2H, m, Lys N—CH₂), 3.95 (1H,m, Lys CH), 4.41 (1H, m, Val CO—CH), 4.48 (2H, d, allyl O—CH₂), 5.06(2H, s, Z CH₂), 5.17 (2H, q, vinyl CH₂), 5.20 (2H, s, PAB CH₂), 5.82(1H, m, vinyl CH), 7.23 and 7.58 (4H, m, PAB Ph), 7.30 (5H, m, Z Ph),7.38 and 8.31 (4H, m, PNP Ph); MS (FAB): 734 (MH⁺), 756 (M+Na)⁺, 772(M+K)⁺; Accurate mass calc. for C₃₇H₄₄N₅O₁₁: 734.3037; found: 734.3036.

EXAMPLE 14 Preparation of Z-Val-N^(ε)-Alloc-Lys-PABC-DOX (14)

Z-Val-N^(ε)-alloc-Lys-PABC-PNP (13) (260.0 mg, 354 μmoles) and DOX-HCl(216 mg, 1.05 equiv.) in NMP (12 ml) at room temperature were treatedwith triethylamine (52 μl). The mixture was allowed to stand in the darkfor 2 days. Workup as described above for Z-Phe-N^(ε)-alloc-Lys-PABC-DOX(8) gave the product as an orange solid (278.0 mg, 69%). ¹H-NMR(CDCl₃/CD₃OD): δ 0.83 (6H, m, Val CH₃), 1.18 (3H, d, sugar CH₃), 1.29,1.41, 1.63 and 1.79 (6H, m, Lys CH₂), 1.72 (2H, m, D-ring CH₂), 1.98(1H, m, Val CH₃—CH), 2.14 (2H, D-ring CH₂), 3.03 (2H, q, sugar CH₂),3.02 (2H, m, Lys N—CH₂), 3.52 (1H, m, sugar HO—CH), 3.76 (1H, m, sugarN—CH), 3.94 (1H, m, Lys CH), 3.99 (3H, s, O—CH₃), 4.39 (1H, m, ValCO—CH), 4.42 (2H, d, allyl O—CH₂), 4.69 (2H, s, PAB CH₂), 4.88 (2H, m, ZCH₂), 5.01 (2H, d, CO—CH ₂—OH), 5.14 (2H, q, vinyl CH₂), 5.18 (1H, m,anomeric CH), 5.41 (1H, br, DOX Ph—CH), 5.80 (1H, m, vinyl CH), 7.13 and7.40 (4H, PAB Ph), 7.26 (5H, s, Z Ph), 7.32, 7.70 and 7.93 (each 1H, m,DOX Ph), 9.25 (1H, br, PAB NH); MS (FAB) 1160 (M+Na)⁺, 1176 (M+K)⁺;Accurate mass calc. for C₅₈H₆₇N₅O₁₉: 1160.4328; found: 1160.4358.

EXAMPLE 15 Preparation of Z-Val-Lys-PABC-DOX-HCl (15)

Z-Val-N^(ε)-alloc-Lys-PABC-DOX (14) (84.3 mg, 74.06 μmoles) in THF (2ml) under argon at room temperature was treated with Pd(PPh₃)₄ (220 μlof a solution of Pd₂dba₃ (4.7 mg, 5.13 μmoles) and PPh₃ (13.5 mg, 10equiv.) in THF (1 ml) under argon), acetic acid (11 μl, 2.5 equiv.) andtributyltin hydride (30 μl, 1.5 equiv.). The mixture was stirred at roomtemperature in the dark for about 1 hour during which time an orangesolid began to form. The mixture was diluted with ether (2 ml) followedby 1M HCl in ether (1 ml) and then more ether (25 ml). The resultingsuspension was sonicated briefly and then filtered. The orange solid waswashed repeatedly with ether and then dissolved in 5:1 CH₂Cl₂/CH₃OH. Tothis was added celite (about 2 g) and the solvents were evaporated. Theresulting solid was dry-loaded atop a celite column (from a slurry in100:1 CH₂Cl₂/CH₃OH). The column was eluted with 1) 100:1 and 2) 10:1CH₂Cl₂/CH₃OH, to give the product as an orange solid (58.5 mg, 72.4%).¹H-NMR (selected peaks)(CDCl₃/CD₃OD): 6 (loss of allyl peaks) 0.83 (6H,m, Val CH₃), 1.20 (3H, d, sugar CH₃), 2.02 (1H, m, Val CH₃—CH), 4.01(3H, s, O—CH₃), 7.10-7.57 (9H, m, Ph), 7.32, 7.72 and 7.91 (each 1H, m,DOX Ph); HPLC (C-18, 15 cm column, 8:2 MeOH/50 mM Et₃N—HCO₂H buffer (pH2.8), 1 ml/min., 495 nm): single peak, retention time 6.1-6.4 min.; MS(FAB)1054 (MH)⁺; Accurate mass calc. for C₅₄H₆₄N₅O₁₇: 1054.4297; found:1054.4283.

EXAMPLE 16 Preparation of Alloc-D-Phe (16)

D-Phe (2.0203 g, 12.29 mmoles) and NaHCO₃ (1.08 g, 1.05 equiv.) in water(30 ml) were treated with diallyl dicarbonate (2.13 ml, 1.05 equiv.) inDME (30 ml). The mixture was stirred at room temperature for about 16hours and then poured into 15% citric acid. The resulting suspension wasextracted with EtOAc (2×100 ml). The combined organic layers were washedwith water (3×100 ml) and brine, dried and evaporated to give acolorless foam which was pure enough to carry on to the next step (3.002g, 98%). ¹H-NMR (CDCl₃/CD₃OD): δ3.13 (2H, AB q, Phe CH₂), 4.52 (2H, d,CH₂—O), 4.64 (1H, q, Phe CH), 5.20 (2H, q, vinyl CH₂), 5.85 (1H, m,vinyl CH), 7.21 (5H, m, Ph); MS (DCI): 250 (MH)⁺, 192 (M—C3H50)⁺;

Anal. calc. for C₁₃H₁₅NO₄—H₂O: C-58.42, H-6.40, N-5.24; Found: C-58.81,H-5.87, N-5.36.

EXAMPLE 17 Preparation of Alloc-D-Phe-NHS (17)

Alloc-D-Phe (16) (3.002 g, 12.04 mmoles) and NHS (1.525 g, 1.1 equiv.)in CH₂Cl₂ at about 0° C. were treated with DCC (2.733 g, 1.1 equiv.).The ice bath was allowed to warm to room temperature and the mixture wasstirred at room temperature for about 16 hours. Workup as describedabove for Z-Phe-NHS (4) gave the product as a colorless foam which wasused without further purification (4.045 g, 97%).

EXAMPLE 18 Preparation of Alloc-D-Phe-Phe (18)

Alloc-D-Phe-NHS (17) (1.7654 g, 5.10 mmoles) in DME (30 ml) at roomtemperature was treated with a solution of Phe (1.263 g, 1.5 equiv.) andNaHCO3 (642.3 mg, 1.5 equiv.) in water (20 ml). The mixture was stirredat room temperature for about 16 hours. The mixture was then poured into15% citric acid (100 ml) and the resulting suspension was extracted withEtOAc (2×100 ml). The combined organic layers were washed with water(3×) and brine, and then dried and evaporated to give a colorless glass.To this was added ether (30 ml) and the mixture was sonicated at roomtemperature for about 15 minutes and then stored in the freezer forabout 1 hour. The solid product was collected by filtration and washedwith ether (1.6973 g, 84%). ¹H-NMR (CDCl₃/CD₃OD): δ2.83-3.16 (4H, m,Ph—CH ₂), 4.45 (2H, d, CH₂—O), 4.63 and 4.89 (each 1H, m, N—CH), 5.21(2H, q, vinyl CH₂), 5.81 (1H, m, vinyl CH), 6.93-7.34 (1OH, m, Ph); MS(DCI): 397 (MH)⁺;

Anal. calc. for C₂₂H₂₄N₂O₅: C-66.65, H-6.10, N-7.07; Found: C-66.42,H-6.19, N-7.09.

EXAMPLE 19 Preparation of Alloc-D-Phe-Phe-NHS (19)

Alloc-D-Phe-Phe (18) (1.0151 g, 2.60 mmoles) and NHS (324.2 mg, 1.1equiv.) in CH₂Cl₂ (25 ml) at 0° C. were treated with DCC (555 mg, 1.05equiv.). The ice bath was allowed to warm to room temperature and themixture was stirred for about 18 hours. The solid DCU was removed byfiltration and the solvent was evaporated. The residue was dissolved inEtOAc and the solution was washed with water (2×) and brine, dried andevaporated to give a white solid which was used without furtherpurification (1.2897 g, 100%).

EXAMPLE 20 Preparation of Alloc-D-Phe-Phe-N^(ε)-Alloc-Lys (20)

Alloc-D-Phe-Phe-NHS (19) (1.2897 g, 2.61 mmoles) in DME (40 ml) wasadded a solution of N^(ε)-alloc-Lys-TFA (945 mg, 1.05 equiv.) and NaHCO₃(461 mg, 2.1 equiv.) in water (20 ml). The mixture was stirredvigorously at room temperature for about 16 hours. Workup as describedabove for Alloc-D-Phe-Phe (18) gave a crude white solid. This wassuspended in ether and alternately sonicated and heated at about 40° C.for several minutes. The mixture was then stored at about 4° C. forabout 2 hours and filtered to remove the white, solid product, which waswashed with cold ether (1.2046 g, 76%). ¹H-NMR (CDCl₃/CD₃OD): δ1.21-1.94 (6H, 4×m, Lys CH₂), 2.79 and 2.91 (each 2H, m, Phe CH₂), 3.08(2H, m, N—CH₂), 4.29 (1H, m, Lys CH), 4.38 and 4.59 (each 1H, m, PheCH), 4.45 and 4.53 (each 2H, d, allyl O—CH₂), 5.20 (4H, m, vinyl CH₂),5.85 (2H, m, vinyl CH), 7.06-7.27 (10H, m, Ph); MS (FAB): 609 (MH)⁺, 631(M+Na)⁺, 647 (M+K)⁺;

Anal. calc. for C₃₂H₄₀N₄O₈: C-63.14, H-6.62, N-9.20; Found: C-63.05,H-6.78, N-9.25.

EXAMPLE 21 Preparation of Alloc-D-Phe-Phe-N^(ε)-Alloc-Lys-PAB-OH (21)

Alloc-D-Phe-Phe-N^(ε)-alloc-Lys (20) (616.8 mg, 1.013 mmoles) andp-aminobenzyl alcohol (137.3 mg, 1.1 equiv.) in THF (12 ml) at roomtemperature were treated with EEDQ (276 mg, 1.1 equiv.). The mixture wasstirred at room temperature for about 18 hours. Workup as describedabove for Z-Phe-Ne-alloc-Lys-PAB-OH (6) gave the product as a whitesolid (685.7 mg, 95%). ¹H-NMR (CDCl₃/CD₃OD): δ1.20-1.98 (6H, 4×m, LysCH₂), 2.95 (4H, m, Phe CH₂), 3.08 (2H, m, N—CH₂), 4.25 (2H, AB q, allylO—CH₂), 4.49 (2H, d, allyl O—CH₂), 4.57 (2H, s, PAB CH₂), 5.15 (4H, m,vinyl CH₂), 5.62 and 5.87 (each 1H, m, vinyl CH), 6.96 and 7.54 (each2H, m, PAB Ph), 7.06-7.31 (1OH, m, Ph); MS (FAB): 714 (MH)⁺, 736(M⁺Na)⁺, 752 (M+K)⁺; Accurate mass calc. for C₃₉H₄₈N₅O₈: 714.3503;found: 714.3494;

Anal. calc. for C₃₉H₄₇N₅O₈-H₂O: C-64.01, H-6.75, N-9.57; Found: C-64.39,H-6.63, N-9.54.

EXAMPLE 22 Preparation of Alloc-D-Phe-Phe-N^(ε)-Alloc-Lys-PABC-PNP (22)

Alloc-D-Phe-Phe-N^(ε)-alloc-Lys-PAB-OH (21) (330.8 mg, 463.4 μmoles) andp-nitrophenyl chloroformate (140.1 mg, 1.5 equiv.) in CH₂Cl₂ (20 ml) atroom temperature were treated with dry pyridine (56.2 μl, 1.5 equiv.).Workup as described above for Z-Phe-Ne-alloc-Lys-PABC-PNP (7) gave theproduct as a white solid (379.0 mg, 93%). ¹H-NMR (CDCl₃/CD₃OD):δ1.20-2.00 (6H, 4×m, Lys CH₂), 2.97 (4H, m, Phe CH₂), 3.10 (2H, m,N—CH₂), 4.21 (2H, AB q, allyl O—CH₂), 4.30, 4.52 and 4.67 (each 1H, m,N—CH), 4.49 (2H, d, allyl O—CH₂), 5.10 (2H, m, vinyl CH₂), 5.22 (2H, s,PAB CH₂), 5.58 and 5.87 (each 1H, m, vinyl CH), 6.93 and 7.66 (each 2H,m, PAB Ph), 7.04-7.25 (1OH, m, Ph), 7.32 and 8.04 (each 2H, m, PNP Ph);MS (FAB): 879 (MH)⁺, 901 (M+Na)⁺, 917 (M+K)⁺; Accurate mass calc. forC₄₆H₅₁N₆O₁₂: 879.3565; found: 879.3533.

EXAMPLE 23 Preparation of Alloc-D-Phe-Phe-N^(ε)-alloc-Lys-PABC-DOX (23)

Alloc-D-Phe-Phe-Ne-alloc-Lys-PABC-PNP (22) (379.0 mg, 431.2 mmoles) andDOX-HCl (262.6 mg, 1.05 equiv.) in NMP (10 ml) at room temperature weretreated with triethylamine (63 ml, 1.05 equiv.). The mixture was storedin the dark at room temperature for 2 days and then diluted with 10%i-propyl alcohol/EtOAc (150 ml). The resulting solution was washed withwater (4×) and brine, filtered to remove a small amount of orange solidby-product, and then evaporated to give an orange solid. This waschromatographed on silica, eluting with 1) 30:1 and 2) 15:1CH₂Cl₂/CH₃OH, to give the product as an orange solid (418.8 mg, 76%).¹H-NMR (CDCl₃/CD₃OD): δ 1.21 (3H, d, sugar CH₃), 1.28-1.96 (6H, 4×m, LysCH₂), 1.76 (2H, m, D-ring CH₂) , 2.18 (D-ring CH₂), 2.87 (2H, m, sugarCH₂), 3.05 (2H, m, N—CH₂), 3.55 (1H, s, sugar HO—CH), 3.78 (1H, m, sugarN—CH), 3.99 (3H, s, CH₃—O), 4.10 (1H, m, sugar CH₃—CH), 4.26 (2H, m,allyl O—CH₂), 4.40 (3H, m, CO—CH), 4.45 (2H, d, allyl O—CH₂), 4.70 (2H,s, CO—CH₂—OH), 4.89 (2H, m, PAB CH₂), 5.16 (4H, m, vinyl CH₂), 5.20 (1H,s, anomeric CH), 5.41 (1H, s, DOX Ph—CH), 5.52 and 5.80 (each 1H, m,vinyl CH), 6.85-7.52 (14H, m, Ph), 7.32, 7.72 and 7.97 (each 1H, m, DOXPh); MS (FAB⁻): 1282.4 (MH)⁻; Accurate mass calc. for C₆₇H₇₄N₆O₂₀Na:1305.4856; found: 1305.4877.

EXAMPLE 24 Preparation of D-Phe-Phe-Lys-PABC-DOX-2HCl (24)

Alloc-D-Phe-Phe-N^(ε)-alloc-Lys-PABC-DOX (23) (164.0 mg, 127.8 μmoles))in degassed 2:1 CH₂Cl₂/CH₃OH (4 ml) at room temperature under argon wastreated with acetic acid (37 μl, 5 equiv.) and then 460 μl of a solutionof Pd(PPh₃)₄ (Pd₂dba₃ (6.4 mg) and PPh₃ (18 mg) in degassed 2:1CH₂Cl₂/CH₃OH (1 ml)). To this was added triethylsilane (61 μl, 3 equiv.)and the mixture was stirred in the dark for about 16 hours at roomtemperature The solvents were removed on the rotovap (40° C.) and theorange, glassy residue was treated with ether (2 ml) and 1M HCl in ether(1 ml). This was sonicated for several minutes. The resulting orangesolid was collected by filtration and then taken up as far as possiblein water. The insoluble material was filtered off and the filtrateevaporated to dryness. The residue was chromatographed on celite,eluting with 1) 50:1, 2) 12:1, and 3) 5:1 CH₂Cl₂/CH₃OH. The firstsolvent system eluted any uncharged material, the second elutedsingly-charged (mono-deprotected) material, and the product eluted inthe third (100.4 mg, 66%). ¹H-NMR (CDCl₃/CD₃OD): δ 1.12 (3H, d, sugarCH₃), 1.00-1.90 (8H, m, Lys CH₂ and D-ring CH₂), 2.07 (2H, m, D-ringCH₂), 2.55-3.16 (8H, m, ⁺H₃N—CH₂ sugar CH₂, Phe CH₂), 3.45 (1H, s, sugarHO—CH), 3.70 (1H, m, sugar N—CH), 3.90 (3H, s, O—CH₃), 4.21, 4.33 and4.43 (each 1H, m, CO—CH), 4.61 (2H, s, CO—CH₂—OH), 4.80 (2H, m, PABCH₂), 5.12 (1H, brs, anomeric CH), 5.33 (1H, brs, DOX Ph—CH), 6.80-7.90(17H, m, Ph); HPLC: (C-18, 15 cm column, 8:2 MeOH/50 mM Et₃N—HCO₂Hbuffer (pH 2.8), 1 ml/min., 495 nm): single peak, retention time 5.5-5.8min.; MS (FAB⁻): 1114.6 (MH)⁻.

EXAMPLE 25 Preparation of Z-Val-Cit (26)

To a solution of Z-Val-NHS (10) (2.98 g, 8.566 mmoles) in DME (25 ml) atroom temperature was added a solution of citrulline (2.25 g, 1.5 equiv.)and NaHCO₃ (1.08 g, 1.5 equiv.) in water (25 ml). The mixture wasstirred vigorously for 2 days. Water (20 ml) containing 2 ml sat. NaHCO₃was added and the mixture was washed with EtOAc and acidified to pH 3with 10% HCl. The resulting suspension was extracted with 10%Bu—OH/EtOAc (3×). The combined organic layers were dried and evaporatedto give a white solid (3.39 g, 97%). ¹H-NMR (CDCl₃/CD₃OD): δ 0.73 (6H,q, Val CH₃), 1.31, 1.46 and 1.63 (4H, m, Cit CH₂), 1.87 (1H, m, ValCH₃—CH), 2.88 (2H, m, N—CH₂), 3.72 (1H, AB q, Cit CH), 4.17 (1H, m, ValCOCH), 4.86 (2H, s, Z CH₂), 7.10 (5H, m, Z Ph); MS (FAB): 409 (MH)⁺, 431(M+Na)⁺, 447 (M+K)⁺; Accurate mass calc. for C₁₉H29N₄O₆: 409.2087;found: 409.2086.

EXAMPLE 26 Preparation of Z-Val-Cit-PAB-OH (27)

Z-Val-Cit (26) (1,0397 g, 2.545 mmoles) and p-aminobenzyl alcohol (470.2mg, 1.5 equiv.) in THF (10 ml) at room temperature were treated withEEDQ (944.2 mg, 1.5 equiv.). The mixture was stirred at room temperaturefor about 16 hours and then diluted with 10% i-Pr—OH/EtOAc (100 ml).This was washed with 10% citric acid, water and brine, dried andevaporated. The pale-yellow solid residue was sonicated in ether for 15min. and the crude solid product was collected by filtration (954.2 mg,73%). ¹H-NMR (CDCl₃/CD₃OD): δ 0.79 (6H, q, Val CH₃), 1.37, 1.53 and 1.72(4H, m, Cit CH₂), 1.92 (1H, m, Val CH₃—CH), 3.00 (2H, m, N—CH₂), 3.85(1H, m, Cit CH), 4.41 (1H, m, Val COCH), 4.45 (2H, s, PAB CH2), 4.95(2H, m, Z CH₂), 7.08-7.40 (9H, m, Ph); MS (FAB): 514 (MH)⁺, 536 (M+Na)⁺,552 (M+K)⁺;

Anal. calc. for C₂₆H₃₅N₅O₆-H₂O: C-58.74, H-7.01, N-13.17; Found:C-59.01, H-6.62, N-13.17.

EXAMPLE 27 Preparation of Z-Val-Cit-PABC-PNP (28)

Z-Val-Cit-PAB-OH (27) (383.0 mg, 745.7 μmoles) and p-nitrophenylchloroformate (225.5 mg, 1.5 equiv.) in THF (10 ml) and CH₂Cl₂ (5 ml)were treated at room temperature with pyridine (91 μl, 1.5 equiv.).Workup as described above for Z-Phe-Ne-alloc-Lys-PABC-PNP (7) gave acrude, pale-yellow solid which was chromatographed on silica, elutingwith 1) 30:1 and 2) 12:1 CH₂Cl₂/CH₃OH, to give the product as anoff-white solid (440.3 mg, 87%). ¹H-NMR (CDCl₃/CD₃OD): δ 0.88 (6H, m,Val CH₃), 1.42, 1.61 and 1.80 (4H, m, Cit CH₂), 2.02 (1H, m, ValCH₃—CH), 3.08 (2H, m, N—CH₂), 3.99 (1H, m, Cit CH), 4.51 (1H, m, ValCOCH), 5.00 (2H, m, Z CH₂), 7.20-7.57 (9H, m, Ph), 7.30 and 8.20 (each2H, m, PNP Ph); MS (FAB): 679 (MH)⁺, 701 (M+Na)⁺, 717 (M+K)⁺; Accuratemass calc. for C₃₃H₃₉N₆O₁₀: 679.2728; found: 679.2720.

EXAMPLE 28 Preparation of Z-Val-Cit-PABC-DOX (29)

Z-Val-Cit-PABC-PNP (28) (126.9 mg, 187 μmoles) and DOX-HCl (119.3 mg,1.1 equiv.) in NMP (5 ml) at room temperature were treated withtriethylamine (29 μl, 1.1 equiv.). The mixture was stirred in the darkat room temperature for 2 days. Workup as described above forAlloc-D-Phe-Phe-N^(ε)-alloc-Lys-PABC-DOX (23) gave a crude orange solid.This was chromatographed on silica, eluting with 1) 12:1, 2) 8:1, and 3)5:1 CH₂Cl₂/CH₃OH, to give the product as a red-orange solid (158.0 mg,78%). ¹H-NMR (CDCl₃/CD₃OD): δ 0.74 (6H, m, Val CH₃), 1.07 (3H, d, sugarCH₃), 1.28-1.88 (4H, m, Cit CH₂), 1.64 and 2.08 (each 2H, m, D-ringCH₂), 1.88 (1H, m, Val CH₃—CH), 2.87 (2H, m, sugar CH₂), 3.42 (1H, brs,sugar HO—CH), 3.95 (1H, m, sugar N—CH), 4.11 (3H, s, O—CH₃), 4.38 (2H,m, CO—CH), 4.58 (2H, s, CO—CH₂—OH), 4.78 (2H, s, PAB CH₂), 4.90 (2H, s,Z CH₂), 5.04 (1H, brs, anomeric CH), 5.30 (1H, brs, DOX Ph—CH),7.00-7.86 (12H, m, Ph), 9.31 (1H, brs, PAB NH); HPLC: (C-18, 15 cmcolumn, 8:2 MeOH/50 mM Et₃N-HCO₂H buffer (pH 2.8), 1 ml/min., 495 nm):single peak, retention time 3.65-3.75 min.; MS, (FAB⁻): 1082.8 (M⁻);Accurate mass calc. for C₅₄H₆₃N₆O₁₈: 1083.4199; found: 1083.4161.

EXAMPLE 29 Preparation of Z-Phe-N^(ε)-alloc-Lys-PABC-2′-Taxol (30)

Taxol (15.8 mg, 18.5 μmoles) and Z-Phe-N^(ε)-alloc-Lys-PABC-PNP (7)(14.5 mg, 1 equiv.) in CH₂Cl₂ (2 ml) at room temperature were treatedwith DMAP (2.5 mg, 1.1 equiv.). After 2 days at room temperature TLC(silica; 25:1 CH₂Cl₂/CH₃OH) indicated completion. EtOAc (25 ml) wasadded and the mixture was washed with 10% citric acid, water, brine,dried and evaporated to give a pale-yellow glass. This waschromatographed on silica, eluting with 30:1 CH₂Cl₂/CH₃OH, to give theproduct as a colorless glass (26.1 mg, 94%). ¹H-NMR (selected peaks): δ1.13, 1.23, 1.68 and 1.81 (each 3H, s, Taxol CH₃), 2.20 and 2.46 (each3H, s, Ac CH₃), 3.13 (2H, m, CON—CH₂), 4.25 (2H, AB q, C-20 CH₂), 4.47(1H, m, C-7 CH), 4.52 (2H, d, alloc O—CH₂), 4.97 (2H, m, Z CH₂), 5.05(2H, s, PAB CH₂), 5.12 (2H, m, vinyl CH₂), 5.45 (1H, d, C-2′ CH), 5.88(1H, m, vinyl CH), 7.10-8.17 (29H, m, Ph), 8.59 (1H, s, PABC NH); MS(Ion spray): 1496.8 (MH)⁺, 1519.6 (M+Na)⁺; Accurate mass calc. forC₈₂H₈₉N₅O₂₂: 1496.6078; found: 1496.6082.

EXAMPLE 30 Preparation of Z-Phe-Lys-PABC-2′-Taxol-HCl (31)

Z-Phe-N^(ε)-alloc-Lys-PABC-2′-Taxol (30) (18.1 mg, 12.09 μmoles) in dryTHF (1 ml) at room temperature under argon was treated with AcOH (1.7μl, 2.5 equiv.), Pd(PPh₃)₄ (45 μl of a solution of Pd₂dba₃ (6.2 mg, 6.77μmoles) and PPh₃ (17.8 mg, 10 equiv.) in dry THF (1 ml)), and Bu₃SnH (5μl, 1.5 equiv.). After about 30 minutes more Bu₃SnH (5 μl) was added.After about 30 more minutes ether (5 ml) and then 1M HCl in ether (1 ml)were added. The resulting suspension was sonicated for several minutesand the white solid was collected by filtration and washed repeatedlywith ether (14.37 mg, 82%). ¹H-NMR (CDCl₃/CD₃OD)(selected peaks): δ(loss of allyl peaks) 2.98 (2H, m, ⁺H₃N—CH₂), 4.27 (2H, AB q, C-20 CH₂),4.39 (1H, m, C-7 CH), 5.02 (2H, m, Z CH₂), 5.09 (2H, m, PAB CH₂),7.06-8.20 (29H, m, Ph); HPLC: (C-18, 15 cm column, 8:2 MeOH/50 mMEt₃N-HCO₂H buffer (pH 2.8), 1 ml/min., 230 nm): single peak, retentiontime 4.8 min., (6:4 MeCN/50 mM Et3N-HCO2H buffer (pH 2.8)): single peak,retention time: 9.6 min.; MS (Ion spray): 1413.2 (MH)⁺; Accurate masscalc. for C₇₈H₈₆N₅O₂₀: 1412.5866; found: 1412.5883.

EXAMPLE 31 Preparation of Boc-Phe-NHS (32)

Boc-Phe (5.4257 g, 20.45 mmoles) and NHS (2.354 g, 1 equiv.) in THF (55ml) at about 0° C. were treated with DCC (4.22 g, 1 equiv.). The icebath was allowed to melt and the mixture was stirred at room temperaturefor about 16 hours. The solid DCU was filtered off and the filtrate wasevaporated to give a white solid which was used without furtherpurification (7.2624 g, 98%). ¹H-NMR: δ 1.39 (9H, s, t-Bu), 2.85 (4H, brs, NHS CH₂), 3.22 (2H, m, Phe CH₂), 4.94 (1H, m, CH), 7.29 (5H, m, Ph).

EXAMPLE 32 Preparation of Boc-Phe-N^(ε)-Fmoc-Lys (33)

N^(ε)-Fmoc-Lys (3.0651 g, 8.32 mmoles) and NaHCO₃ (769 mg, 1.1 equiv.)in water (50 ml) and DME (20 ml) were treated, at room temperature, witha solution of Boc-Phe-NHS (32) (3.015 g, 1 equiv.) in DME (40 ml). Themixture was stirred vigorously at room temperature for about 18 hoursand then diluted with EtOAc (100 ml) and 10% citric acid. The aqueouslayer was re-extracted with EtOAc (50 ml). The combined organic layerswere washed with water (2×) and brine, dried and evaporated to give apale-yellow solid. This was dissolved in ether and a small amount ofundissolved solid was removed by filtration. The filtrate was evaporatedto dryness and the pale-yellow foamy residue was dried in vacuo (5.0881g, 99%). ¹H-NMR (CDCl₃/CD₃OD): δ 1.30, 1.48, 1.67 and 1.85 (6H, m, LysCH₂), 1.35 (9H, s, t-Bu), 3.01 (2H, m, Phe CH₂), 3.12 (2H, m, N—CH₂),4.18 (1H, t, Fmoc CH), 4.36 (2H, d, Fmoc CH₂), 4.41 and 4.50 (each 1H,m, CO—CH), 7.12-7.77 (13H, m, Ph); MS (FAB): 616 (MH)⁺, 638 (M+Na)⁺, 654(M+K)⁺;

Anal. calc. for C₃₅H₄₁N₃O₇: C-68.27, H-6.71, N-6.82; Found: C-68.13,H-6.84, N-6.44.

EXAMPLE 33 Preparation of Boc-Phe-N^(ε)-Fmoc-Lys-PAB-OH (34)

Boc-Phe-N^(ε)-Fmoc-Lys (33) (4.8207 g, 7.83 mmoles) and p-aminobenzylalcohol (1.061 g, 1.1 equiv.) in THF (50 ml) at room temperature weretreated with EEDQ (2.13 g, 1.1 equiv.). The mixture was stirred at roomtemperature for about 16 hours. Workup as described above forZ-Phe-N^(ε)-alloc-Lys-PAB-OH (6) gave the product as an off-white solid(4.4579 g, 79%). ¹H-NMR (CDCl₃/CD₃OD): δ 1.28, 1.48, 1.63 and 1.84 (6H,m, Lys CH₂), 1.33 (9H, s, t-Bu), 3.00 (2H, m, Phe CH₂), 3.11 (2H, m,N—CH₂), 4.15 (1H, t, Fmoc CH), 4.31 (2H, d, Fmoc CH₂), 4.38 (2H, m,CO—CH), 4.57 (2H, s, PAB CH₂), 7.08-7.75 (17H, m, Ph); MS (FAB): 721(MH)⁺, 743 (M+Na)⁺, 759 (M+K)⁺;

Anal. calc. for C₄₂H₄₈N₄O₇-½ H₂O: C-69.12, H-6.77, N-7.68; Found:C-68.96, H-6.87, N-7.64.

EXAMPLE 34 Preparation of 2′-Fmoc-Taxol (35)

Taxol (134.6 mg, 157.6 μmoles) and Fmoc-NHS (58.5 mg, 1.1 equiv.) inCH₂Cl₂ (3 ml) at room temperature were treated with DMAP (19.3 mg, 1equiv.). After about 5 days at room temperature TLC (silica; 25:1CH₂Cl₂/CH₃OH) indicated completion. EtOAc (50 ml) was added and themixture was washed with 10% citric acid, water, brine, dried andevaporated. The residue was chromatographed on silica, eluting with 35:1CH₂Cl₂/CH₃OH, to give the product as a colorless glass (165.6 mg, 98%).¹H-NMR: δ 1.13, 1.24 and 1.67 (each 3H, s, C-16, C-17 and C-19 CH₃),1.92 (3H, s, C-18 CH₃), 1.87 and 2.52 (2H, m, C-6 CH₂), 2.22 and 2.44(each 3H, s, Ac CH₃), 2.41 (2H, m, C-14 CH₂), 2.50 (1H, d, C-7 OH), 3.82(1H, d, C-3 CH), 4.28-4.51 (6H, m, C-20 CH₂, C-7 CH, Fmoc CH and CH₂),4.98 (1H, d, C-5 CH), 5.47 (1H, d, C-2′ CH), 5.69 (1H, d, C-2 CH), 6.03(1H, m, C-3′ CH), 6.30 (1H, s, C-10 CH), 6.32 (1H, t, C-13 CH), 6.99(1H, d, NH), 7.22-8.20 (23H, m, Ph); MS (FAB): 1076 (MH)⁺, 1098 (M+Na)⁺,1114 (M+K)⁺; Accurate mass calc. for C₂₆H₆₂NO₁₆: 1076.4069; found:1076.4031.

EXAMPLE 35 Preparation of Boc-Phe-N^(ε)-Fmoc-Lys-PABC-7-Taxol-2′-Fmoc(36)

2′-Fmoc-taxol (35) (112.1 mg, 90.3 μmoles) in dry CH₂Cl₂ (1 ml) underargon at about 0° C. was treated with pyridine (8 μl, 1.1 equiv.) anddiphosgene (6.5 μl, 0.6 equiv.). After about 40 minutesBoc-Phe-N^(ε)-Fmoc-Lys-PAB-OH (65.1 mg, 1 equiv.) and DMAP (0.5 mg) inCH₂Cl₂ (1 ml)/pyridine (0.2 ml) were added. The mixture was stirred atabout 0° C. for about 30 minutes and then at room temperature for about4 hours. EtOAc (30 ml) was then added and the solution was washed with10% citric acid (2×), water and brine, then dried and evaporated to givea white solid. This was chromatographed on silica, eluting with 30:1CH₂Cl₂/CH₃OH, to give the product as a colorless glass (81.7 mg, 50%,two of the three product-containing fractions were contaminated with2′-Fmoc-taxol)). ¹H-NMR (CDCl₃/CD₃OD): δ 1.19, 1.22 and 1.80 (each 3H,s, C-16, C-17 and C-19 CH₃), 1.10-1.90 (6H, m, Lys CH₂), 1.38 (9H, s,t-Bu), 1.82 and 2.54 (each 1H, m, C-6 CH₂), 2.05 (3H, s, C-18 CH₃), 2.23and 2.42 (each 1H, m, C-14 CH₂), 2.18 and 2.47 (each 3H, s, Ac CH₃),3.09 (2H, m, Phe CH₂), 3.19 (2H, m, Lys N—CH₂), 3.98 (1H, d, C-3 CH),4.15-4.52 (7H, m, Phe and Lys CO—CH, Fmoc CH₂ and CH, C-20 CH₂), 4.98(1H, m, C-5 CH), 5.14 (2H, m, PAB CH₂), 5.48 (1H, d, C-2′ CH), 5.55 (1H,m, C-7 CH), 5.69 (1H, m, C-2 CH), 6.02 (1H, m, C-3′ CH), 6.29 (1H, m,C-13 CH), 6.41 (1H, s, C-10 CH), 6.96-8.18 (40H, m, Ph); MS (FAB): 1823(MH)⁺, 1846 (M+Na)⁺, 1862 (M+K)⁺.

EXAMPLE 36 Preparation of Boc-Phe-Lys-PABC-7-Taxol-HCl (37)

Boc-Phe-N^(ε)-Fmoc-Lys-PABC-7-Taxol-2′-Fmoc (36) (74.6 mg, 40.95 μmoles)in THF (2 ml) at room temperature was treated with 2% DBU in THF (2 ml).After about 6 minutes at room temperature ether (25 ml) was added andthe resulting white solid was collected by filtration and washed withether. The solid was suspended in ether (5 ml) and treated with 1M HClin ether (2 ml). After about 2 minutes the solid was filtered off andwashed thoroughly with ether. The solid was chromatographed on LH-20lipophilic sephadex, eluting with 1:1 CH₂Cl₂/CH₃OH, to give the productas a colorless glasss (35.6 mg, 90%). ¹H-NMR (CDCl₃/CD₃OD): δ 1.13, 1.19and 1.78 (each 3H, s, C-16, C-17 and C-19 CH₃), 1.37 (9H, s, t-Bu),1.10-1.90 (6H, m, Lys CH₂), 1.86 and 2.54 (each 1H, m, C-6 CH₂), 2.05(3H, s, C-18 CH₃), 2.16 and 2.38 (each 3H, s, Ac CH₃), 2.97 (2H, m,⁺H₃N—CH₂), 3.12 (2H, m, Phe CH₂), 3.90 (1H, d, C-3 CH), 4.24 (2H, m,C-20 CH₂), 4.45 and 4.68 (each 1H, m, Phe and Lys CO—CH), 4.83 (1H, brs,C-2′ CH), 4.91 (1H, d, C-5 CH), 5.12 (2H, m, PAB CH₂), 5.48 (1H, m, C-7CH), 5.67 (1H, d, C-2 CH), 5.78 (1H, d, c-3′ CH), 6.12 (1H, m, C-13 CH),6.33 (1H, s, C-10 CH), 7.08-8.12 (24H, m, Ph); HPLC: (C-18, 15 cmcolumn, 8:2 MeOH/50 mM Et₃N—HCO₂H buffer (pH 2.8), 1 ml/min., 230 nm):single peak, retention time: 7.1-7.3 min.; MS (Ion spray): 1379.2 (MH)⁺;Accurate mass calac. for C₇₅H₈₈N50₂₀: 1378.6023; found: 1378.6043.

EXAMPLE 37 Preparation of Boc-Phe-N^(ε)-Fmoc-Lys-PABC-Cl (38)

Boc-Phe-N^(ε)-Fmoc-Lys-PAB-OH (34) (211.2 mg, 293 μmoles) in pyridine(0.5 ml) and CH₂Cl₂ (2 ml) at −42° C. (dry ice-MeCN) under argon wastreated with diphosgene (21.2 μl, 0.6 equiv.). The mixture was stirredat about −42° C. for about 20 minutes during which time solid pyridiniumhydrochloride had precipitated out of solution. This solution was usedimmediately.

EXAMPLE 38 Preparation of Boc-Phe-N^(ε)-Fmoc-Lys-PABC-MMC (39)

To the above solution of Boc-Phe-N^(ε)-Fmoc-Lys-PABC-Cl (38) at about−42° C. was added MMC (118.0 mg, 1.2 equiv.) in NMP (1 ml). The coolingbath was allowed to warm to room temperature gradually and the mixturewas stirred in the dark for about 12 hours at room temperature. Themixture was diluted with 10% i-Pr-OH/EtOAc (50 ml) and 10% citric acid(50 ml). The organic layer was washed with water (3×) and brine, driedand evaporated to give a purple-brown residue. This was chromatographedon a 1 mm silica prep. plate, eluting with 12:1 CH₂Cl₂/CH₃OH, to givethe product as a light purple solid (108.0 mg, 34%). ¹H-NMR(CDCl₃/CD₃OD): δ 1.21, 1.43, 1.61 and 1.81 (6H, m, Lys CH₂), 1.32 (9H,s, t-Bu), 2.10 (3H, s, MMC CH₃), 2.99 (2H, m, Phe CH₂), 3.11 (2H, m, LysN—CH₂), 3.14 (3H, s, O—CH₃), 3.20-3.50 (3H, m, C-1 and C-2 CH, and C-3CH), 3.62 (1H, ABq, C-9 CH), 4.18 (1H, t, Fmoc CH), 4.22 and 4.89 (each1H, ABq, C-10 CH₂), 4.32 (2H, d, Fmoc CH₂), 4.41 (1H, d, C-3 CH), 4.45(2H, m, Phe and Lys CO—CH), 5.01 (2H, m, PAB CH₂), 7.05-7.90 (17H, m,Ph); MS (FAB): 1082 (MH)⁺, 1103 (M+Na)⁺, 1119 (M+K)⁺; Accurate masscalc. for C₅₈H₆₄N₈O₁₃Na: 1103.4491; found: 1103.4451.

EXAMPLE 39 Preparation of Boc-Phe-Lys-PABC-MMC-HCl (40)

Boc-Phe-N^(ε)-Fmoc-Lys-PABC-MMC (39) (11.2 mg, 10.36 μmoles) in THF (1ml) at room temperature was treated with 2% DBU in THF (1 ml). A finepurple solid slowly formed. After about 5 minutes the volume was reducedto about 1 ml on the rotovap (bath temp. 30° C.) and ether (10 ml) wasadded. The resulting solid was collected by filtration and washed withether. The solid was suspended in ether (2 ml) and treated with 1M HClin ether (3 ml). After about 2 minutes the solid was filtered off,washed thoroughly with ether, and then triturated with CH₂Cl₂ (2 ml).The resulting solid was collected by filtration and washed with CH₂Cl₂(9.1 mg, 98%). ¹H-NMR (CDCl₃/CD₃OD): δ 1.30 (9H, s, t-Bu), 1.20-1.90(6H, m, Lys CH₂), 1.94 (3H, s, MMC CH₃), 2.83 (2H, m, ⁺H₃N—CH₂), 2.98(2H, m, Phe CH₂), 3.13 (3H, s, O—CH₃), 3.20-3.70 (4H, m, C-1 and C-2 CH,C-3 CH and ABq, C-9 CH), 4.14 and 4.82 (each 1H, ABq, C-10 CH),4.25-4.52 (3H, m, Phe and Lys CO—CH and C-3 CH), 4.97 (2H, m, PAB CH₂),7.12 (5H, brs, Phe Ph), 7.23 and 7.50 (each 2H, m, PAB Ph); HPLC: (C-18,15 cm column, 65:35 MeOH/50 mM Et₃N—HCO₂H buffer (pH 2.8), 1 ml/min.,365 nm): single peak, retention time: 4.1-4.3 min.; MS (FAB): 859 (MH)⁺,881 (M+Na)⁺, 897 (M+K)⁺; Accurate mass calc. for C₄₃H₅₅N₈O₁₁: 859.3990;found: 859.3980.

EXAMPLE 40 Preparation of N^(α)-Fmoc-N^(ε)-Mtr-Lys (41)

N^(α)-Fmoc-Lys (14.840 g, 40.28 mmoles) was suspended in dry CH₂Cl₂ (200ml) at room temperature under argon. Trimethylsilyl chloride (10.9 ml, 2equiv.) was added with vigorous stirring, and the mixture was heated atreflux for about one hour, and then cooled to about 0° C. DIEA (14.0 ml,2 equiv.) was added, followed by p-anisyldiphenylmethyl chloride (13.061g, 1.05 equiv.) in CH₂Cl₂ (50 ml). The ice bath was removed and themixture was stirred for about 2 hours at room temperature. Methanol (8.2ml, 5 equiv.) was added and stirring was continued for one hour and thenthe solvents were evaporated. The residue was partitioned between ethylacetate and pH 5 buffer (biphthalate). The organic layer was washed withwater and brine, dried and evaporated, giving a pale orange gum. Thiswas flushed with CH₂Cl₂ and dried in vacuo to give a foam which wascarried on without further purification (25.693 g, 99%). ¹H-NMR (CDCl₃)δ 1.26 and 1.68 (2H and 4H, m, Lys CH₂), 2.45 (2H, m, N—CH₂), 3.71 (3H,s, OCH₃), 4.05-4.40 (4H, m, Fmoc CH₂ and CH, CO—CH), 6.81 (2H, d, MeOPho-CH), 7.15-7.77 (20H, m, Ph); MS (FAB) 641 (MH)⁺, 663 (M+Na)⁺, 679(M+K)⁺.

EXAMPLE 41 Preparation of N^(ε)-Mtr-Lys (42)

N^(α)-Fmoc-N^(ε)-Mtr-Lys (41) (10.006 g, 15.615 mmoles) in CH₂Cl₂ (50ml) at room temperature was treated with diethylamine (40 ml). Themixture was stirred at room temperature for about 24 hours and then thesolvents were evaporated and the residue flushed with CH₂Cl₂ (3×100 ml).The pale yellow residue was triturated with ether. The resultingsuspension was sonicated for several minutes, and the solid wascollected by filtration, washed with ether and dried in vacuo forseveral hours (6.191 g, 95%). ¹H-NMR (CDCl₃/CD₃OD) δ 1.34, 1.57 and 1.72(6H, m, Lys CH₂), 2.05 (2H, m, N—CH₂), 3.38 (1H, m, CO—CH), 3.68 (3H, s,OCH₃), 3.71 (2H, d, MeOPh o-CH), 7.03-7.40 (12H, m, Ph); MS (FAB) 419.2(MH)⁺, 441.4 (M+Na)⁺, 457.4 (M+K)⁺.

EXAMPLE 42 Preparation of Fmoc-Phe-NHS (43)

Fmoc-Phe (5.1043 g, 13.17 mmoles) and NHS (1.592 g, 1.05 equiv.) inCH₂Cl₂ (100 ml) at about 0° C. were treated with DCC (2.854 g, 1.05equiv.). The ice bath was allowed to warm to room temperature and themixture was stirred for about 14 hours. The DCU by-product was removedby filtration and the filtrate was evaporated. The resulting crudeproduct, a colorless glass, was used without further purification.

EXAMPLE 43 Preparation of Fmoc-Phe-N^(ε)-Mtr-Lys (44)

A suspension of N^(ε)-Mtr-Lys (42) (4.686 g, 11.20 mmoles) and NaHCO₃(941.0 mg, 1 equiv.) in water (100 ml) and DME (50 ml) was treated witha solution of Fmoc-Phe-NHS (43) (11.20 mmoles) in DME (50 ml). THF (25ml) was then added to aid solubility. The mixture was stirred at roomtemperature for 2 days and then as much DME as possible was removed onthe rotovap (bath at about 30° C.). The resulting gummy suspension waspartitioned between ethyl acetate and pH 5 buffer. The organic phase waswashed with water and brine, dried and evaporated to give a pale yellowfoam. This was flushed with CH₂Cl₂ (100 ml). TLC showed the product tobe fairly pure and it was carried on without further purification (8.559g, 97%). ¹H-NMR (CDCl₃/CD₃OD) δ 1.10-1.93 (6H, m, Lys CH₂), 2.31 (2H, t,N—CH₂), 3.00 (2H, m, Phe CH₂), 3.71 (3H, s, O—CH₃), 4.02-4.48 (5H, m,Fmoc CH₂ and CH, CO—CH), 6.79 (2H, d, MeOPh o-CH), 7.00-7.75 (25H, m,Ph); MS (FAB) 788.2 (MH)⁺, 810.4 (M+Na)⁺, 826 (M+K)⁺;

Anal. calc. for C₅₀H₄₉N₃O₆-H₂O: C-74.51, H-6.38, N-5.21; Found: C-74.17,H-6.57, N-5.41.

EXAMPLE 44 Preparation of Fmoc-Phe-N^(ε)-Mtr-Lys-PAB-OH (45)

Fmoc-Phe-N^(ε)-Mtr-Lys (44) (7.728 g, 9.808 mmoles) and p-aminobenzylalcohol (1.450 g, 1.2 equiv.) in CH₂Cl₂ (100 ml) at room temperaturewere treated with EEDQ (3.640 g, 1.5 equiv.). The mixture was stirred atroom temperature for about 20 hours and then the solvent was evaporated(water bath at about 30° C.). The solid residue was triturated withether (200 ml) and the resulting suspension sonicated for about 15minutes and left to stand at room temperature for about 2 hours. Theresulting solid was collected by filtration, washed well with ether, anddried in vacuo (7.6140 g, 87%). ¹H-NMR (CDCl₃/CD₃OD) δ 0.98-1.91 (6H, m,Lys CH₂), 2.06 (2H, t, N—CH₂), 2.97 (2H, m, Phe CH₂), 3.71 (3H, s,O—CH₃), 4.12 (1H, t, Fmoc—CH), 4.20-4.41 (4H, m, Fmoc CH₂ and CO—CH),4.59 (2H, s, PAB CH₂), 6.72 (2H, d, MeOPh o-CH), 7.00-7.73 (29H, m, Ph);MS (FAB) 891.4 (MH)⁺, 916.7 (M+Na)⁺, 931 (M+K)⁺;

Anal. calc. for C₅₇H₅₆N₄O₆-H₂O: C-75.14, H-6.42, N-6.15; Found: C-75.25,H-6.02, N-6.49.

EXAMPLE 45 Preparation of Phe-N^(ε)-Mtr-Lys-PAB-OH (46)

Fmoc-Phe-N^(ε)-Mr-Lys-PAB-OH (45) (4.2857 g, 4.80 mmoles) in CH₂Cl₂ (35ml) at room temperature was treated with diethylamine (50 ml). Themixture was sonicated briefly and stirred at room temperature for 4hours after which time no starting material was observed by TLC. Thesolvents were evaporated and the residue was flushed with CH₂Cl₂ andchromatographed on silica, eluting with 1) 2% methanol/CH₂Cl₂, 2) 3%methanol/CH₂Cl₂, and 3) 4% methanol/CH₂Cl₂, to give the product as acolorless foam (2.230 g, 69%). ¹H-NMR (CDCl₃) δ 1.26-2.00 (6H, m, LysCH₂), 2.12 (2H, t, N—CH₂), 2.75 and 3.21 (each 1H, ABq, Phe CH₂), 3.68(1H, ABq, Phe CO—CH), 3.76 (3H, s, O—CH₃), 4.42 (1H, q, Lys CO—CH), 4.66(2H, brs, PAB CH₂), 6.79 (2H, d, MeOPh o-CH), 7.10-7.42 (21H, m, Ph),7.81 (1H, d, amide NH), 8.71 (1H, s, PAB NH); MS (FAB) 693.4 (M+Na)⁺,709 (M+K)⁺;

Anal. calc. for C₄₂H₄₆N₄O₄-½H₂O: C-74.20, H-6.97, N-8.24; Found:C-74.28, H-7.00, N-8.34.

EXAMPLE 46 Preparation of MC-Phe-N^(ε)-Mtr-Lys-PAB-OH (47)

Phe-N^(ε)-Mtr-Lys-PAB-OH (46) (448.1 mg, 0.668 mmoles) and DIEA (0.128ml, 1.1 equiv.) in CH₂Cl₂ (5 ml) at room temperature were treated withMC-NHS (230.4 mg, 1.12 equiv.) in CH₂Cl₂ (2 ml). The mixture was stirredat room temperature for 3 days. Ethyl acetate (60 ml) was added and themixture was washed with pH 5 buffer (2×), water and brine, dried andevaporated. The residue was triturated with ether (60 ml) and theresulting solid collected by filtration and washed with ether (563.8 mg,98%). ¹H-NMR (CDCl₃) δ 1.05-1.96 (12H, m, Lys and caproyl CH₂), 2.07(2H, t, Lys N—CH₂), 2.18 (2H, t, CO—CH₂), 3.02 (2H, m, Phe CH₂), 3.39(2H, t, M—CH₂), 3.71 (3H, s, O—CH₃), 4.64 (3H, s and m, PAB CH₂ and LysCO—CH), 4.99 (1H, q, Phe CO—CH), 6.61 (2H, s, M CH), 6.71 (2H, d, MeOPho-CH), 6.89 (1H, m, amide NH), 7.00-7.55 (21H, m, Ph), 8.97 (1H, brs,PAB NH); MS (FAB) 864 (MH)⁺, 886 (M+Na)⁺, 902.4 (M+K)⁺.

EXAMPLE 47 Preparation of MC-Phe-N^(ε)-Mtr-Lys-PABC-PNP (48)

MC-Phe-N^(ε)-Mtr-Lys-PAB-OH (47) (679.3 mg, 0.786 mmoles) andbis-p-nitrophenyl carbonate (1.196 g, 5 equiv.) under argon at roomtemperature were dissolved in CH₂Cl₂ (25 ml) and treated with DIEA(0.411 ml, 3 equiv.). After 3 days TLC indicated completion. The volumewas reduced to about 5 ml on the rotovap and the residue was dilutedwith ethyl acetate (80 ml) and washed with pH 5 buffer, water and brine,dried and evaporated. The resulting solid was triturated with ether (80ml), and the solid was collected by filtration, washed with ether, andchromatographed on silica, eluting with 1) 1:1 and 2) 8:1 ethylacetate/hexane (the sample was loaded on the column in a minimum amountof 8:1 ethyl acetate/hexane), to give the product as a pale yellow glass(670.7 mg, 83%). ¹H-NMR (CDCl₃) δ 1.10-1.95 (12H, m, Lys and caproylCH₂), 2.04 (2H, t, Lys N—CH₂), 2.13 (2H, t, CO—CH₂), 3.04 (2H, m, PheCH₂), 3.39 (2H, t, M—CH₂), 3.72 (3H, s, O—CH₃), 4.58 (1H, q, Lys CO—CH),4.86 (1H, q, Phe CO—CH), 5.27 (2H, s, PAB CH₂), 6.58 (1H, d, amide NH),6.61 (2H, s, M CH), 6.72 (2H, d, MeOPh o-CH), 7.03-7.62 (27H, m, Ph andNH), 8.22 (2H, d, PNP CH), 8.86 (1H, brs, PAB NH); MS (FAB) 1029 (MH)⁺,1051.5 (M+Na)⁺, 1069.4 (M+K)⁺.

EXAMPLE 48 Preparation of MC-Phe-N^(ε)-Mtr-Lys-PABC-DOX (49)

MC-Phe-N^(ε)-Mtr-Lys-PABC-PNP (48) (126.6 mg, 0.123 mmoles) and DOX.HCl(71.3 mg, 1 equiv.) in NMP (5 ml) at room temperature were treated withDIEA (21.4 μl, 1 equiv.). After 2 days standing in the dark at roomtemperature the mixture was diluted with ethyl acetate (60 ml) andwashed with water (4×) and brine, dried and evaporated. The residue waschromatographed on silica, eluting with 1) 25:1 and 2) 20:1CH₂Cl₂/methanol, to give the product as an orange glass (149.0 mg, 85%).¹H-NMR (CDCl₃) δ 1.10-1.95 (14H, m, Lys and caproyl CH₂, D-ring CH₂),1.27 (3H, d, sugar CH₃), 2.10 (4H, m, Lys N—CH₂ and caproyl CO—CH₂),2.23 (2H, m, D-ring CH₂), 3.03 (2H, m, Phe CH₂), 3.20 (2H, m, sugarCH₂), 3.41 (2H, t, M—CH₂), 3.67 (1H, brs, sugar HO—CH), 3.77 (3H, s, MtrO—CH₃), 4.08 (3H, s, DOX O—CH₃), 4.13 (sugar N—CH), 4.40 (1H, m, PheCO—CH), 4.56 (2H, m, Lys CO—CH and sugar CH₃—CH), 4.76 (2H, brs,CO—CH₂—OH), 4.99 (2H, m, PAB CH₂), 5.29 (1H, brs, anomer CH), 5.51 (1H,brs, DOX Ph—CH), 5.18, 6.02 and 6.38 (each 1H, m, NH), 6.62 (2H, s, MCH), 6.77 (2H, d, MeOPh o-CH), 7.00-7.60 (22H, m, Ph), 7.78 and 8.03(each 1H, m, DOX Ph CH), 8.22 (1H, brs, PAB NH); MS (FAB) 1433.8 (MH)⁺,1456.0 (M+Na)⁺, 1471.8 (M+K)⁺.

EXAMPLE 49 Preparation of MC-Phe-Lys-PABC-DOX.Cl₂CHCO₂H (50)

A stirred solution of MC-Phe-N^(ε)-Mtr-Lys-PABC-DOX (49) (1.1520 g,0.804 mmoles) in CH₂Cl₂ (50 ml) and anisole (8.73 ml, 100 equiv.) wastreated with dichloroacetic acid (0.663 ml, 10 equiv.). After about 1hour ethyl acetate (80 ml) was added and the resulting suspension wasstored in the freezer for about 1.5 hours. The solid was collected byfiltration, washed with ethyl acetate, and dried in vacuo. The filtratewas concentrated to about 30 ml on the rotovap (bath at about 27° C.)and then ether (50 ml) was added. The resulting suspension was stored inthe freezer for about 1 hour and then filtered. The orange solid wastriturated repeatedly with CH2Cl2 and then dried in vacuo (1.0092 g,97%). ¹H-NMR (CDCl₃/CD₃OD) δ 1.10-1.90 (14H, m, Lys and caproyl CH₂,D-ring CH₂), 1.21 (3H, d, sugar CH₃), 2.10 (2H, t, caproyl CO—CH₂), 2.20(2H, m, D-ring CH₂), 2.88 (2H, m, Lys N—CH₂), 3.02 (2H, m, Phe CH₂),3.12 (2H, m, sugar CH₂), 3.38 (2H, t, M—CH₂), 3.52 (1H, brs, sugarHO—CH), 3.79 (1H, m, sugar HN—CH), 4.02 (3H, s, DOX O—CH₃), 4.10 (1H, m,sugar CH₃—CH), 4.43 and 4.54 (each 1H, m, Phe and Lys CO—CH), 4.72 (2H,s, DOX CO—CH₂—OH), 4.92 (2H, m, PAB CH₂), 5.24 (1H, br s, anomeric CH),5.44 (1H, br s, DOX Ph—CH—O-sugar), 5.84 (1H, s, Cl₂CH), 6.67 (2H, s, MCH), 7.10 (5H, brs, Phe Ph), 7.21 and 7.48 (each 2H, d, PAB Ph), 7.38,7.77 and 7.99 (each 1H, d, t, and d, resp., DOX Ph); HPLC: (C-18, 15 cmcolumn, 8:2 methanol/50 mM triethylammonium formate buffer (pH 2.8), 1ml/min., 495 nm): single peak, retention time: 4.4-4.5 min.; MS (FAB⁻):1159 (M−H)⁻; Accurate mass calc. for C₆₀H₆₈N₆O₁₈Na: 1183.4488; found:1183.4457.

EXAMPLE 50 Preparation of MC-Phe-N^(ε)-Mtr-Lys-PABC-MMC (51)

A stirred mixture of MC-Phe-N^(ε)-Mtr-Lys-PABC-PNP (48) (160.4 mg,0.1559 mmoles), HOBt (211.0 mg, 10 equiv.) and MMC (57.3 mg, 1.1 equiv.)in NMP (5 ml) at room temperature was treated with DIEA (0.271 ml, 10equiv.). After about 14 hours at room temperature ethyl acetate (100 ml)was added and the mixture was washed with pH 5 buffer, water and brine,dried and evaporated. The residue was chromatographed on silica, elutingwith 1) 25:1 and 2) 20:1 CH₂Cl₂/methanol, to give the product as apurple glass (136.2 mg, 71%). ¹H-NMR (CDCl₃) δ 1.08-1.90 (12H, m, CH₂),1.73 (3H, s, MMC CH₃), 2.10 (4H, m, Lys N—CH₂ and CO—CH₂), 3.05 (2H, m,Phe CH₂), 3.18 (3H, s, MMC O—CH3), 3.23-3.50 (5H, m, C-1, C-2 and C-3 CHand M—CH₂), 3.63 (1H, ABq, C-9 CH), 3.74 (3H, s, Mtr O—CH₃), 4.28 and4.90 (each 1H, t and ABq, C-10 CH₂), 4.41 (2H, d and m, C-3 CH and PheCO—CH), 4.71 (1H, m, Lys CO—CH), 5.01 (2H, m, PAB CH₂), 5.09 (1H, brs,amide NH), 5.30 (4H, brs, NH₂), 6.31 and 6.88 (each 1H, d, amide NH),6.63 (2H, s, M CH), 6.76 (2H, d, MeOPh o-CH), 7.06-7.57 (21H, m, Ph),8.81 (1H, brs, PAB NH); MS (FAB) 1246.5 (M+Na)⁺, 1262.3 (M+K)⁺.

EXAMPLE 51 Preparation of MC-Phe-Lys-PABC-MMC.CLCH₂CO₂H (52)

A stirred solution of MC-Phe-N^(ε)-Mtr-Lys-PABC-MMC (51) (68.1 mg, 55.6μmoles) in CH₂Cl₂ (3 ml) and anisole (0.604 ml, 100 equiv.) was treatedwith chloroacetic acid (1M in CH₂Cl₂, 0.56 ml, 10 equiv.). A purpleprecipitate gradually formed. After 3 hours ether (5 ml) was added. Theresulting solid was collected by filtration and washed with ether andCH₂Cl₂, and then dissolved in methanol. HPLC showed it to be >95% pure(44.7 mg, 74%). ¹H-NMR (CDCl₃/CD₃OD) δ 1.11, 1.40, 1.63 and 1.77 (12H,m, CH₂), 2.09 (2H, t, CO—CH₂), 3.02 (2H, m, Phe CH₂), 3.13 (3H, s, MMCO—CH₃), 3.23-3.50 (5H, m, C-1, C-2 and C-3 CH and M—CH₂), 3.56 (1H, ABq,C-9 CH), 3.92 (2H, brs, ClCH₂), 4.13 and 4.82 (each 1H, t and ABq, C-10CH₂), 4.30 (1H, d, C-3 CH), 4.41 (1H, m, Phe CO—CH), 4.65 (1H, m, LysCO—CH), 4.99 (2H, q, PAB CH₂), 6.63 (2H, s, M CH), 7.10 (5H, brs, PhePh), 7.22 and 7.48 (each 2H, d, PAB Ph); MS (FAB) 952.3 (MH)⁺, 974(M+Na)⁺, 990.3 (M+K)⁺; HPLC: (C-18, 15 cm column, 65:35 methanol/50 mMtriethylammonium formate buffer (pH 2.8), 1 ml/min., 360 nm): singlepeak, retention time: 2.84 min.

EXAMPLE 52 Preparation of 2′-Methoxytrityl-Taxol (53)

A stirred solution of taxol (0.51 g, 0.597 mmoles) and p-methoxytritylchloride (4.63 g, 25 equiv.) in CH₂Cl₂ (14 ml) under nitrogen at roomtemperature was treated with pyridine (1.23 ml, 25 equiv.). After about16 hours at room temperature the solvent was evaporated and the residuedissolved in ethyl acetate. The solution was washed with cold pH 5buffer (2×100 ml), water and brine, dried and evaporated. The residuewas chromatographed on silica, eluting with 3% methanol/CH₂Cl₂, to givethe product as a white solid (482 mg, 72%). ¹H NMR (CDCl₃) δ 1.11, 1.17and 1.55 (each 3H, s, C-16, C-17 and C-19 CH₃), 1.67 (3H, s, C-18 CH₃),1.90 and 2.54 (2H, m, C-6 CH₂), 2.26 and 2.51 (each 3H, s, Ac CH₃), 2.54(2H, m, C-14 CH₂), 3.66 (1H, d, C-3 CH), 3.78 (3H, s, O—CH₃), 4.21 (2H,ABq, C-20 CH₂), 4.41 (1H, m, C-7 CH), 4.63 (1H, d, C-2′ CH), 4.92 (1H,d, C-5 CH), 5.62 (1H, d, C-2 CH), 5.70 (2H, m, C-13 and C-3′ CH), 6.22(1H, s, C-10 CH), 6.74 (2H, d, MeOPh o-CH), 7.09-7.60 (23H, m, Ph), 7.80and 8.09 (each 2H, d, Bz o-CH); MS (FAB) 1148 (M+Na)⁺, 1164 (M+K)⁺.

EXAMPLE 53 Preparation of MC-Phe-N^(ε)-Mtr-Lys-PABC-7-Taxol-2′-Mtr (54)

2′-Methoxytrityl-taxol (53) (218.8 mg, 0.194 mmoles) in dry CH₂Cl₂ (3ml) under argon at about 0° C. was treated with DIEA (34 μl, 1 equiv.),pyridine (15.7 μl, 1 equiv.) and then diphosgene (12 μl, 0.5 equiv.).The ice bath was removed and the mixture was stirred at room temperaturefor about 1 hour and then recooled to about 0° C.MC-Phe-N^(ε)-Mtr-Lys-PAB-OH (47) (167.9 mg, 1 equiv.) was flushed withdry CH₂Cl₂ (6 ml), dried in vacuo and then dissolved in dry CH₂Cl₂ (2ml) and DIEA (34 μl, 1 equiv.). This solution was added via syringe tothe crude chloroformate at about 0° C. After about 10 minutes the icebath was removed and the mixture was stirred at room temperature forabout 18 hours. The mixture was diluted with ethyl acetate and washedwith pH 5 buffer, water and brine, dried and evaporated. The residue waschromatographed on silica, eluting with 1) 2:1 CH₂Cl₂/ethyl acetate, 2)1:1 ethyl acetate/CH₂Cl₂, 3) 4:1 ethyl acetate/CH₂Cl₂ and 4) ethylacetate, to give the product as a colorless glass (237.9 mg, 61%), alongwith unreacted starting materials. ¹H NMR (CDCl₃) δ 1.13, 1.16 and 1.57(each 3H, s, C-16, C-17 and C-19 CH₃), 1.10-1.80 (12H, m, Lys andcaproyl CH₂), 1.88 and 2.61 (each 1H, m, C-6 CH₂), 1.78 (3H, s, C-18CH₃), 2.10 (4H, m, Lys N—CH₂ and caproyl CO—CH₂), 2.17 and 2.29 (each3H, s, Ac CH₃), 3.06 (2H, m, Phe CH₂), 3.42 (2H, t, caproyl N—CH₂), 3.75and 3.78 (each 3H, s, O—CH₃), 3.82 (1H, m, C-3 CH), 4.21 (2H, ABq, C-20CH₂), 4.42 and 4.70 (each 1H, q, Phe and Lys CO—CH), 4.62 (1H, d, C-2′CH), 4.93 (1H, d, C-5 CH), 5.19 (2H, q, PAB CH₂), 5.59 (1H, m, C-7 CH),5.62 (1H, d, C-2 CH), 5.72 (2H, m, C-3′ CH and C-13 CH), 6.17 and 6.60(each 1H, brd, amide NH), 6.32 (1H, s, C-10 CH), 6.64 (2H, s, M CH),6.77 (4H, m, MeOPh o-CH), 7.05-7.62 (44H, m, Ph), 7.80 and 8.06 (each2H, d, Bz o-CH), 8.37 (1H, brs, PAB NH).

EXAMPLE 54 Preparation of MC-Phe-Lys-PABC-7-Taxol.ClCH₂CO₂H (55)

A stirred solution of MC-Phe-N^(ε)-Mtr-Lys-PABC-7-Taxol-2′-Mtr 54 (194.8mg, 0.097 mmoles) in CH₂Cl₂ (4.5 ml) and anisole (1.05 ml, 100 equiv.)was treated with chloroacetic acid (1M in CH₂Cl₂, 0.97 ml, 10 equiv.).After about 4 hours ether (25 ml) was added. The resulting solid wascollected by filtration and washed with ether (142.0 mg, 94%). ¹H NMR(CDCl₃) δ 1.13, 1.20 and 1.72 (each 3H, s, C-16, C-17 and C-19 CH₃),1.10-1.90 (12H, m, Lys and caproyl CH₂), 2.13 and 2.33 (each 3H, s, AcCH₃), 2.96 (2H, m, ⁺H₃N—CH₂), 3.05 (2H, m, Phe CH₂), 3.38 (2H, m,caproyl N—CH₂), 3.86 (1H, d, C-3 CH), 4.21 (2H, m, C-20 CH₂), 4.50 and4.61 (each 1H, m, Phe and Lys CO—CH), 4.77 (1H, brs, C-2′ CH), 4.91 (1H,d, C-5 CH), 5.10 (2H, m, PAB CH₂), 5.42 (1H, m, C-7 CH), 5.64 (1H, d,C-2 CH), 5.71 (1H, m, C-3′ CH), 6.11 (1H, m, C-13 CH), 6.30 (1H, s, C-13CH), 6.73 (2H, s, M CH), 7.00-8.20 (24H, m, Ph); HPLC (C-18, 15 cmcolumn, 7:3 acetonitrile/50 mM triethylammonium formate buffer (pH 2.8),1 ml/min., 250 nm): single peak, retention time 2.91 min.; MS (FAB)1471.6 (MH⁺), 1509.5 (M+Na)⁺, 1511.8 (M+K)⁺.

EXAMPLE 55 Preparation of Fmoc-Val-NHS (56)

Fmoc-Val (5.060 g, 14.91 mmoles) and NHS (1.72 g, 1 equiv.) in THF (50ml) at about 0° C. were treated with DCC (3.080 g, 1 equiv.). Themixture was stirred at room temperature for about 16 hours and then thesolid DCU by-product was filtered off and washed with THF. The solventwas removed on the rotovap and the resulting colorless, glassy solid wasused without purification in the next step.

EXAMPLE 56 Preparation of Fmoc-Val-Cit (57)

Fmoc-Val-NHS (56) (14.91 mmoles) in DME (40 ml) was added to a solutionof L-citrulline (2.743 g, 1.05 equiv.) and NaHCO₃ (1.315 g, 1.05 equiv.)in water (40 ml). THF (20 ml) was added to aid solubility, and themixture was stirred at room temperature for about 16 hours. Aqueouscitric acid (15%, 75 ml) was added and the mixture was extracted with10% isopropanol/ethyl acetate (2×100 ml). The solid product began toprecipitate but remained with the organic layer. The suspension waswashed with water (2×150 ml) and the solvents were evaporated. Theresulting white solid was dried in vacuo for about 5 hours and thentreated with ether (80 ml). After sonication and trituration the whitesolid product was collected by filtration (5.8007 g, 78%). ¹H-NMR(DMSO-d₆) δ 0.87 (6H, q, Val CH₃), 1.40, 1.59 and 1.69 (4H, m, Cit CH₂),1.97 (1H, m, Val CH₃—CH), 2.94 (2H, q, Cit N—CH₂), 3.92 (1H, t, FmocCH), 4.10-4.35 (2H, m, Val and Cit CO—CH), 4.23 (2H, t, Fmoc CH₂), 5.37(2H, brs, Cit NH₂), 5.92 (1H, t, Cit NH), 7.28-7.90 (8H, m, Ph), 8.15(1H, d, amide NH); MS (FAB) 497 (MH)⁺, 519 (M+Na)⁺, 535 (M+K)⁺; Accuratemass calc. for C₂₆H₃₃N₄O₆: 497.2400; found: 497.2394;

Anal. calc. for C₂₆H₃₂N₄O₆: C-62.89, H-6.50, N-11.28; Found: C-62.92,H-6.67, N-11.07.

EXAMPLE 57 Preparation of Fmoc-Val-Cit-PAB-OH (58)

Fmoc-Val-Cit (57) (1.0443 g, 2.103 mmoles) and p-aminobenzyl alcohol(518.0 mg, 2 equiv.) in 2:1 CH₂Cl₂/methanol (35 ml) were treated withEEDQ (1.0402 g, 2 equiv.). The mixture was stirred in the dark at roomtemperature for 1.5 days. The solvents were removed on the rotovap (bathtemp. about 40° C.) and the white solid residue was triturated withether (75 ml). The resulting suspension was sonicated for about 5minutes and then left to stand for about 30 minutes. The solid wascollected by filtration and washed repeatedly with ether (1.0070 g,80%). ¹H-NMR (DMSO-d₆) δ 0.88 (6H, t, Val CH₃), 1.41 and 1.65 (4H, m,Cit CH₂), 2.00 (1H, m, Val CH₃—CH), 2.99 (2H, m, Cit N—CH₂), 3.92 (1H,t, Fmoc CH), 4.24 (2H, d, Fmoc CH₂), 4.19-4.50 (2H, m, Val and CitCO—CH), 4.43 (2H, d, PAB CH₂), 5.11 (1H, t, PAB OH), 5.42 (2H, brs, CitNH₂), 5.98 (1H, t, Cit NH), 7.15-7.92 (12H, m, Ph), 8.12 (1H, d, amideNH), 9.99 (1H, brs, PAB NH); MS (FAB) 602 (MH)⁺, 624 (M+Na)⁺, 640(M+K)⁺; Accurate mass calc. for C₃₃H₄₀N₅O₆: 602.2979; found: 602.2977;

Anal. calc. for C₃₃H₃₉N₅O₆: C-65.87, H-6.53, N-11.64; Found: C-65.61,H-6.49, N-11.73.

EXAMPLE 58 Preparation of Val-Cit-PAB-OH (59)

Fmoc-Val-Cit-PAB-OH (58) (245.2 mg, 407.5 μmoles) in NMP (4 ml) at roomtemperature was treated with diethylamine (0.8 ml). The mixture was leftto stand at room temperature for about 16 hours and then the solventswere removed on the rotovap (bath temp about 40° C.). The thick, oilyresidue was treated with CH₂Cl₂ (15 ml). With scraping and sonicationthe first-formed gum became a solid which was collected by filtrationand washed with CH₂Cl₂ (141.6 mg, 92%). ¹H-NMR (DMSO-d₆) δ 0.82 (6H,2×d, Val CH₃), 1.39, 1.59 and 1.66 (4H, m, Cit CH₂), 1.92 (1H, m, ValCH₃—CH), 2.98 (1H, m, Val CO—CH), 3.03 (2H, d, Val NH₂), 4.45 (2H, d,PAB CH₂), 4.48 (1H, m, Cit CO—CH), 5.10 (1H, brt, PAB OH), 5.41 (2H,brs, Cit NH₂), 5.99 (1H, brt, Cit NH), 7.21 and 7.52 (each 2H, d, PABPh), 8.12 (1H, brd, amide NH), 10.03 (1H, brs, PAB NH); MS (FAB) 380(MH)⁺, 402 (M+Na)⁺, 418 (M+K)⁺.

EXAMPLE 59 Preparation of MC-Val-Cit-PAB-OH (60)

Val-Cit-PAB-OH (59) (136.8 mg, 360.5 μmoles) and MC-NHS (122.3 mg, 1.1equiv.) in NMP (5 ml) at room temperature were left to stand for about16 hours. The NMP was removed on the rotovap (bath temp. about 40° C.)and the thick, oily residue was triturated with ether (20 ml). The solidproduct was collected by filtration and washed repeatedly with ether(205.7 mg, 99.6%). ¹H-NMR (DMSO-d₆) δ 0.82 (6H, ABq, Val CH₃), 1.10-1.90(10H, m, Cit and caproyl CH₂), 1.92 (1H, m, Val CH₃—CH), 2.16 (2H, t,caproyl CO—CH₂), 2.98 (2H, m, Cit N—CH₂), 3.33 (2H, t, M—CH₂), 4.19 (1H,t, Val CO—CH), 4.38 (1H, m, Cit CO—CH), 4.42 (2H, brd, PAB CH₂), 5.10(1H, brt, PAB OH), 5.42 (2H, brs, Cit NH₂), 5.97 (1H, brt, Cit NH), 6.99(2H, s, M CH), 7.21 and 7.52 (each 2H, d, PAB Ph), 7.82 and 8.07 (each1H, d, amide NH), 9.90 (1H, brs, PAB NH); MS (FAB) 573 (MH)⁺, 595(M+Na)⁺, 611 (M+K)⁺; Accurate mass calc. for C₂₈H₄₁N₆O₇: 573.3037;found: 573.3016.

EXAMPLE 60 Preparation of MC-Val-Cit-PABC-PNP (61)

MC-Val-Cit-PAB-OH (60) (112.4 mg, 196.3 μmoles) under argon at roomtemperature was dissolved in dry pyridine (3 ml). The solution wascooled to about 0° C. and p-nitrophenyl chloroformate (119 mg, 3 equiv.)in CH₂Cl₂ (2 ml) was added all at once. After about 10 minutes at about0° C. the ice bath was removed and the mixture was stirred at roomtemperature for about 2 hours. Ethyl acetate (50 ml) and 15% citric acid(75 ml) were added. The organic phase was washed with more citric acid,water and brine, dried and evaporated to give a light-yellow gum. Thiswas chromatographed on silica, eluting with 1) 20:1 and 2) 15:1CH₂Cl₂/methanol, to give the product as a white solid (21.5 mg, 15%).¹H-NMR (CDCl₃/CD₃OD) δ 0.90 (6H, d, Val CH₃), 1.16-1.95 (10H, m, Cit andcaproyl CH₂), 2.12 (1H, m, Val CH₃—CH), 2.23 (2H, t, caproyl CO—CH₂),3.17 (2H, m, Cit N—CH₂), 3.48 (2H, t, M—CH₂), 4.20 (1H, m, Val CO—CH),4.59 (1H, m, Cit CO—CH), 5.22 (2H, s, PAB CH₂), 6.66 (2H, s, M CH), 6.91and 7.79 (each 1H, d, amide NH), 7.34 and 7.60 (each 2H, d, PAB Ph),7.34 and 8.23 (each 2H, d, PNP Ph), 9.49 (1H, brs, PAB NH); MS (FAB) 738(MH)⁺, 760 (M+Na)⁺, 776 (M+K)⁺.

EXAMPLE 61 Preparation of MC-Val-Cit-PABC-DOX (62)

MC-Val-Cit-PABC-PNP (61) (21.2 mg, 28.7 μmoles) and DOX.HCl (18.3 mg,1.1 equiv.) in NMP (1.5 ml) at room temperature were treated withdiisopropylethylamine (5.5 μl, 1.1 equiv.). The mixture was left tostand in the dark at room temperature for 2 days and then CH₂Cl₂ (25 ml)was added. A fine precipitate formed. The suspension was stored in thefreezer overnight and then the orange solid was collected by filtrationand washed with CH₂Cl₂. TLC showed some product remaining in the motherliquors along with most of the close-moving impurities. The crude solidwas chromatographed on silica, eluting with 1) 15:1, 2) 10:1 and 3) 5:1CH₂Cl₂/methanol (the sample was loaded in a minimum amount of 2:1CH₂Cl₂/methanol), to give the product as an orange solid (22.4 mg, 68%).¹H-NMR (CDCl₃/CD₃OD) δ 0.83 (6H, d, Val CH₃), 1.18 (3H, d, sugar CH₃),1.20-1.86 (12H, m, Cit and caproyl CH₂, D-ring CH₂), 1.93 (1H, m, ValCH₃—CH), 2.12 (2H, m, D-ring CH₂), 2.17 (2H, t, caproyl CO—CH₂),2.90-3.20 (4H, q and m, sugar CH₂ and Cit N—CH₂), 3.39 (2H, t, M—CH₂),3.50 (1H, brs, HO—CH), 3.98 (3H, s, O—CH₃), 4.02 (1H, m, Val CO—CH),4.05 (1H, m, sugar CH₃—CH), 4.46 (1H, m, Cit CO—CH), 4.68 (2H, s,CO—CH₂—OH), 4.88 (2H, q, PAB CH₂), 5.16 (1H, brs, anomeric CH), 5.39(1H, brs, DOX Ph—CH), 6.62 (2H, s, M CH), 7.13 and 7.42 (each 2H, d, PABPh), 7.32, 7.71 and 7.92 (each 1H; d, t and d; DOX Ph); MS (FAB) 1141(M)⁺, 1164.6 (M+Na)⁺, 1180 (M+K)⁺; Accurate mass calc. forC₅₆H₆₇N₇O₁₉Na: 1164.4389; found: 1164.4363.

EXAMPLE 62 Preparation of N-Boc-aminocaproic acid (63)

6-Aminocaproic acid (5.2331 g, 39.89 mmoles) and NaHCO₃ (3.3514 g, 1equiv.) in water (50 ml) were treated with d-t-butyl dicarbonate (9.58g, 1.1 equiv.). The mixture was stirred at room temperature overnightand then water (150 ml) and sat. NaHCO₃ (5 ml) were added. The solutionwas extracted with ether (100 ml) and then solid citric acid (10 g) wasadded, giving an oily suspension. This was extracted with Ethyl acetate(3×). The combined organic phases were washed with water and brine,dried and evaporated to give a colorless oil which solidified undervacuum (9.23 g, quant.). ¹H-NMR (DMSO-d₆) δ 1.10-1.55 (6H, m, caproylCH₂), 1.33 (9H, s, CH₃), 2.28 (2H, m, CO—CH₂), 2.88 (2H, m, N—CH₂), 6.77(1H, m, NH); MS (DCI) 232 (MH)⁺, 176 (MH-C₄H₉)⁺;

Anal. calc. for C₁₁H₂₁NO₄: C-57.12, H-9.15, N-6.06; Found: C-57.11,H-9.22, N-6.09.

EXAMPLE 63 Preparation of Boc-NH-C-NHS (64)

N-Boc-aminocaproic acid (63) (9.23 g, 39.9 mmoles) and NHS (5.05 g, 1.1equiv.) in THF (75 ml) at room temperature were treated with DCC (9.05g, 1.1 equiv.). The mixture was stirred at room temperature for about 16hours and then the solid DCU by-product was filtered off. The filtratewas evaporated to give a thick oil which was dissolved in CH₂Cl₂ (150ml). After standing for about 1 hour more DCU was filtered off. Thefiltrate was again evaporated and the thick oily residue dried in vacuoupon which it gradually solidified. The product was used without furtherpurification (13.052 g, 99.6%).

EXAMPLE 64 Preparation of Boc-NH-C-Phe (65)

A solution of Boc-NH-C-NHS (64) (12.52 g, 38.13 mmoles) in DME (100 ml)was added to a solution of L-Phe (6.930 g, 1.1 equiv.) and NaHCO₃ (3.524g, 1.1 equiv.) in water (100 ml) at room temperature THF (30 ml) wasadded to increase solubility. The mixture was stirred at roomtemperature for about 16 hours and then 15% citric acid (100 ml) wasadded. The suspension was extracted with 10% isopropanol/ethyl acetate(3×80 ml) and the combined organic phases were washed with water andbrine, dried and evaporated to give a white solid. This was trituratedwith ether and the resulting white solid was collected by filtration andwashed with ether (12.122 g, 84%). ¹H-NMR (DMSO-d₆) δ 1.09 and 1.30 (6H,m, caproyl CH₂), 1.38 (9H, s, CH₃), 1.80-2.25 (2H, m, CO—CH₂), 2.82 (4H,m, Phe CH₂ and N—CH₂), 4.52 (1H, m, CO—CH), 6.73 (1H, m, NH), 7.20 (5H,m, Ph); MS (DCI) 379 (MH)⁺, 323 (MH-C₄H₉)⁺, 279 (MH-C₅H₉O₂)⁺;

Anal. calc. for C₂₀H₃₀N₂O₅: C-63.47, H-7.99, N-7.40; Found: C-63.37,H-8.05, N-7.76.

EXAMPLE 65 Preparation of Boc-NH-C-Phe-NHS (66)

Boc-NH-C-Phe (65) (11.527 g, 30.46 mmoles) and NHS (3.86 g, 1.1 equiv.)in THF (100 ml) at about 0° C. were treated with DCC (6.913 g, 1.1equiv.). The mixture was stirred at room temperature for about 16 hoursand worked up as described above for Boc-NH-C-NHS (64) to give theproduct as a colorless glass which was used without further purification(14.369 g, 99.2%).

EXAMPLE 66 Preparation of Boc-NH-C-Phe-N-Fmoc-Lys (67)

Boc-NH-C-Phe-NHS (66) (14.369 g, 30.22 mmoles) in DME (100 ml) was addedto a solution of N^(ε)-Fmoc-Lys (11.222 g, 1 equiv.) and NaHCO₃ (2.560g, 1 equiv.) in water (50 ml) and DME (50 ml). The mixture was stirredvigorously at room temperature for about 16 hours and then 15% citricacid (150 ml) and 10% isopropanol/ethyl acetate (250 ml) were added. Theaqueous phase was extracted with more 10% isopropanol/ethyl acetate(2×100 ml). The combined organic phases were washed with water andbrine, dried and evaporated to give an off-white solid. This wastriturated with ether and the white, solid product was collected byfiltration and washed with ether (17.842 g, 81%). ¹H-NMR (CDCl₃/CD₃OD) δ1.00-1.92 (12H, m, Lys and caproyl CH₂), 1.42 (9H, s, CH₃), 2.09 (2H, m,CO—CH₂), 2.96 (2H, m, Phe CH₂), 3.10 (2H, m, caproyl N—CH₂), 3.31 (2H,m, Lys N—CH₂), 4.18 (1H, t, Fmoc CH), 4.37 (2H, d, Fmoc CH₂), 4.46 and4.71 (each 1H, m, Phe and Lys CO—CH), 7.10-7.80 (13H, m, Ph); MS (FAB)729 (MH)⁺, 751 (M+Na)⁺, 767 (M+K)⁺;

Anal. calc. for C₄₁H₅₂N₄O₈—H₂O: C-65.93, H-7.32, N-7.66; Found: C-66.07,H-7.32, N-7.66.

EXAMPLE 67 Preparation of Boc-NH-C-Phe-N^(ε)-Fmoc-Lys-PAB-OH (68)

Boc-NH-C-Phe-N^(ε)-Fmoc-Lys (67) (15.716 g, 21.56 mmoles) andp-aminobenzyl alcohol (3.983 g, 1.5 equiv.) in THF (100 ml) at roomtemperature were treated with EEDQ (8.000 g, 1.5 equiv.). The mixturewas stirred at room temperature for about 16 hours and then evaporatedto dryness (water bath temperature 30° C.). The residue was trituratedwith ether (100 ml) and the white, solid product was collected byfiltration and washed with ether (16.453 g, 92%). ¹H-NMR (DMSO-d₆) δ0.90-1.80 (12H, m, Lys and caproyl CH₂), 1.35 (9H, s, CH₃), 2.00 (2H, t,CO—CH₂), 2.66-3.07 (6H, m, N—CH₂ and Phe CH₂), 4.19 (1H, m, Fmoc CH),4.23 (2H, d, Fmoc CH₂), 4.36 and 4.58 (each 1H, m, Phe and Lys CO—CH),4.41 (2H, s, PAB CH₂), 7.10-8.22 (17H, m, Ph), 9.94 (1H, brs, PAB NH);MS (FAB) 834 (MH)⁺, 856 (M+Na)⁺, 872 (M+K)⁺;

Anal. calc. for C₄₈H₅₉N₅O₈-½H₂O: C-68.39, H-7.17, N-8.31; Found:C-68.18, H-7.12, N-8.42.

EXAMPLE 68 Preparation of MC-NH-C-Phe-N^(ε)-Fmoc-Lys-PAB-OH (69)

Boc-NH-C-Phe-N^(ε)-Fmoc-Lys-PAB-OH (60) (2.1323 g, 2.860 mmoles) wasdissolved in 2:1 CH₂Cl₂/TFA (30 ml). The mixture was sonicated at roomtemperature for about 15 minutes and then left to stand for about 1hour. The solvents were evaporated and the residual brown oil was driedin vacuo for about 1 hour. Ether (75 ml) was added and the oil wasscraped until it solidified. The solid was collected by filtration,washed with ether and dried in vacuo for several hours. It was thendissolved in 3:1 DME/water (40 ml) and treated with a solution of MC-NHS(788.2 mg, 1 equiv.) in DME (20 ml) and solid NaHCO₃ (540 mg, 2.5equiv.). The mixture was stirred at room temperature for about 16 hours.As much DME as possible was removed on the rotovap (water bath temp.about 30° C.), leaving a gummy solid (which eventually solidified) inwater. The solid was filtered, washed with water and dried in vacuo. Itwas then triturated with ether (25 ml) and the solid product wascollected by filtration and washed with ether (1.4283 g, 60%). ¹H-NMR(CDCl₃/CD₃OD) δ 1.00-1.90 (18H, m, Lys and caproyl CH₂), 2.07 (4H, m,Phe CH₂ and CO—CH₂), 2.22 (2H, t, CO—CH₂), 3.05 (4H, m, Lys N—CH₂ andcaproyl N—CH₂), 3.41 (2H, m, M—CH₂), 4.11 (1H, t, Fmoc CH), 4.28 (2H, d,Fmoc CH₂), 4.38 and 4.63 (each 1H, m, Phe and Lys CO—CH), 4.52 (2H, s,PAB CH₂), 5.61 (2H, s, M CH), 6.96-7.71 (17H, m, Ph); MS (FAB) 927.5(MH)⁺, 949.3 (M+Na)⁺, 965.3 (M+K)⁺; Accurate mass calc. for C₅₃H₆₃N₆O₉:927.4657; found: 927.4642.

EXAMPLE 69 Preparation of MC-NH-C-Phe-N^(ε)-Fmoc-Lys-PABC-PNP (70)

MC-NH-C-Phe-N^(ε)-Fmoc-Lys-PAB-OH (69) (1.3783 g, 1.487 mmoles) andp-nitrophenyl chloroformate (449.5 mg, 1.5 equiv.) in CH₂Cl₂ (50 ml) atroom temperature were treated with pyridine (0.18 ml, 1.5 equiv.). Thesuspension was sonicated at room temperature for about 30 minutes andthen stirred for about 16 hours. More p-nitrophenyl chloroformate (150mg, 0.5 equiv.) and pyridine (0.06 ml, 0.5 equiv.) were added and themixture was again sonicated for about 30 minutes and stirred for about 4hours. Workup as described above for MC-Val-Cit-PABC-PNP (61) gave thecrude product as a gummy solid. This was chromatographed on silica,eluting with 1) 35:1, 2) 25:1 and 3) 20:1 CH₂Cl₂/methanol, to give theproduct as a pale-yellow, gummy solid (593.1 mg, 0.543 mmoles). ¹H-NMR(CDCl₃/CD₃OD) δ 1.10-1.95 (18H, m, Lys and caproyl CH₂), 2.12 (4H, m,caproyl CO—CH₂), 3.00 (2H, m, Phe CH₂), 3.11 (4H, m, Lys and caproylN—CH₂), 3.44 (2H, t, M—CH₂), 4.13 (1H, t, Fmoc CH), 4.32 (2H, d, FmocCH₂), 4.39 and 4.63 (each 1H, m, Phe and Lys CO—CH), 5.18 (2H, s, PABCH₂), 6.63 (2H, s, M CH), 7.00-8.25 (21H, m, Ph); MS (FAB): 1114(M+Na)⁺, 1130 (M+K)⁺; Accurate mass calc. for C₆₀H₆₆N₇O₁₃: 1092.4719;found: 1092.4680.

EXAMPLE 70 Preparation of MC-NH-C-Phe-N^(ε)-Fmoc-Lys-PABC-DOX (71)

MC-NH-C-Phe-N^(ε)-Fmoc-Lys-PABC-PNP (70) (382.8 mg, 0.350 mmoles) andDOX.HCl (213 mg, 1.05 equiv.) in NMP (16 ml) were treated withdiisopropylethylamine (61 μl, 1 equiv.). The mixture was allowed tostand in the dark for 2 days. Workup as described above forMC-Val-Cit-PABC-DOX (62) gave the product as an orange glass (293.1 mg,56%). ¹H-NMR (CDCl₃/CD₃OD) δ 1.00-1.85 (20H, m, Lys and caproyl CH₂,D-ring CH₂), 1.21 (3H, d, sugar CH₃), 2.09 (4H, m, caproyl CO—CH₂), 2.17(2H, m, D-ring CH₂), 2.80-3.27 (8H, m, Lys and caproyl N—CH₂, sugar CH₂,Phe CH₂), 3.40 (2H, t, M—CH₂), 3.53 (1H, brs, HO—CH), 3.78 (1H, m, sugarN—CH), 3.99 (3H, s, O—CH₃), 4.11 (2H, t, Fmoc CH and sugar CH₃—CH), 4.29(2H, d, Fmoc CH₂), 4.33 and 4.57 (each 1H, m, Phe and Lys CO—CH), 4.71(2H, s, CO—CH₂—OH), 4.89 (2H, q, PAB CH₂), 5.20 (1H, brs, anomeric CH),5.42 (1H, brs, DOX Ph—CH), 6.60 (2H, s, M CH), 6.90-8.00 (20H, m, Ph);MS (FAB) 1519 (M+Na)⁺, 1534 (M+K)⁺; Accurate mass calc. forC₈₁H₈₉N₇O₂₁Na: 1518.6009; found: 1518.5962.

EXAMPLE 71 Preparation of MC-NH-C-Phe-Lys-PABC-DOX.HCl (72)

MC-NH-C-Phe-N^(ε)-Fmoc-Lys-PABC-DOX (71) (95.2 mg, 63.6 μmoles) in NMP(0.3 ml) was diluted with THF (10 ml) and then, with stirring, treatedwith 2% DBU in THF (10 ml). After about 45 seconds ether (40 ml) wasadded and the resulting blue solid was collected by filtration andwashed with ether. The solid was resuspended in ether (10 ml) andtreated with 1M HCl in ether (10 ml). After several minutes the orangesolid was filtered off, washed repeatedly with ether and triturated withCH₂Cl₂ (25 ml). The resulting orange-red solid was collected byfiltration and chromatographed on LH-20 lipophilic SEPHADEX, elutingwith 1:1 CH₂Cl₂/methanol. The product-containing fractions were combinedand re-chromatographed on LH-20, eluting with methanol, to give theproduct as an orange glass, with minor contaminents as shown by HPLC(40.2 mg, 48.2%). ¹H-NMR (CDCl₃/CD₃OD) δ (selected peaks) 1.00-1.95(23H, m, sugar CH₃, Lys and caproyl CH₂, D-ring CH₂), 2.00-2.40 (6H, m,caproyl CO—CH₂ and D-ring CH₂), 2.96 (2H, m, ⁺H₃N—CH₂), 4.05 (3H, s,O—CH₃), 4.72 (2H, s, CO—CH₂—OH), 4.93 (2H, brs, PAB CH₂), 5.17 (1H, brs,anomeric CH), 5.42 (1H, brs, DOX Ph—CH), 6.63 (2H, brs, M CH), 6.90-8.20(12H, m, Ph).

EXAMPLE 72 Preparation of Fmoc-Phe-N^(ε)-Mtr-Lys-NHS (73)

A stirred mixture of Fmoc-Phe-N^(ε)-Mtr-Lys (44) (1.8873 g, 2.40 mmoles)and NHS (303.2 mg, 1.1 equiv.) in CH₂Cl₂ (40 ml) at about 0° C. wastreated with DCC (543.6 mg, 1.1 equiv.). After about 24 hours at roomtemperature the DCU was filtered off and the filtrate evaporated and theresidue taken up in ethyl acetate. This was washed with water (2×) andbrine, dried and evaporated. The residue was chromatographed on silica,eluting with 1:1 ethyl acetate/hexane. Much of the product decomposed onthe column (472.5 mg, 22%). ¹H-NMR (CDCl₃) δ 1.00-1.98 (6H, m, CH₂),2.01 (2H, t, N—CH₂), 2.77 (4H, brs, NHS CH₂), 3.09 (2H, m, Phe CH₂),3.76 (3H, s, O—CH₃), 4.10-4.51 (4H, m, Fmoc CH₂ and CH, Phe CO—CH), 4.83(1H, m, Lys CO—CH), 5.48 and 6.41 (each 1H, m, NH), 6.79 (2H, d, MeOPho-CH), 7.06-7.80 (25H, m, Ph).

EXAMPLE 73 Preparation of Fmoc-Phe-N^(ε)-Mtr-Lys-GABA (74)

A solution of Fmoc-Phe-N^(ε)-Mtr-Lys-NHS (73) (472.5 mg, 0.534 mmoles)in DME (25 ml) was added to a stirred solution of GABA (83 mg, 1.5equiv.) and NaHCO₃ (67 mg, 1.5 equiv.) in water (15 ml) at roomtemperature. After 16 hours at room temperature as much DME as possiblewas removed on the rotovap and the resulting suspension was partitionedbetween ethyl acetate and pH 5 buffer. The organic phase was washed withwater and brine, dried and evaporated. The residue was triturated withether and the resulting white solid collected by filtration (387.0 mg,83%). ¹H-NMR (CDCl₃) δ 0.96-1.99 (8H, m, CH₂), 2.10-2.42 (4H, m, LysN—CH₂ and CO—CH₂), 3.03 (2H, m, Phe CH₂), 3.22 (2H, m, GABA N—CH₂),4.03-4.66 (5H, m, Fmoc CH₂ and CH, CO—CH), 6.78 (2H, d, MeOPh o-CH),7.00-7.77 (25H, m, Ph); MS (FAB) 895 (M+Na)⁺, 911 (M+K)⁺.

EXAMPLE 74 Preparation of Fmoc-Phe-N^(ε)-Mtr-Lys-GABA-MMC (75)

A stirred mixture of Fmoc-Phe-N-Mtr-Lys-GABA (74) (296.9 mg, 0.340mmoles), HOBt (46 mg, 1 equiv.) and MMC (119.4 mg, 1.05 equiv.) in NMP(3 ml) and CH₂Cl₂ (3 ml) at room temperature was treated with DCC (77.2mg, 1.1 equiv.). After about 14 hours at room temperature ethyl acetatewas added and the solution was washed with water (3×) and brine, driedand evaporated. The residue was chromatographed on silica, eluting with25:1 CH₂Cl₂/methanol, to give the product as a purple glass (303.1 mg,75%). ¹H-NMR (CDCl₃) δ 0.97-1.90 (8H, m, CH₂), 1.71 (3H, s, MMC CH₃),2.08 (2H, m, Lys N—CH₂), 2.46 (2H, m, CO—CH₂), 2.99 (2H, m, Phe CH₂),3.12 (2H, m, GABA N—CH₂), 3.20 (3H, s, MMC O—CH₃), 3.28-3.55 (3H, m,C-1, C-2 and C-3 CH), 3.68 (1H, ABq, C-9 CH), 3.73 (3H, s, Mtr O—CH₃),4.04-4.51 and 4.64 (7H, m, Fmoc CH₂ and CH, C-10 CH₂, CO—CH), 5.14 (2H,br, NH₂), 5.38, 5.49, 5.70 and 6.67 (each 1H, br, NH), 6.79 (2H, d,MeOPh o-CH), 7.03-7.78 (25H, m, Ph); MS (FAB) 1189.8 (MH)⁺, 1211(M+Na)⁺, 1227.5 (M+K)⁺.

EXAMPLE 75 Preparation of Phe-N^(ε)-Mtr-Lys-GABA-MMC (76)

Fmoc-Phe-N^(ε)-Mtr-Lys-GABA-MMC (75) (236.1 mg, 0.198 mmoles) in CH₂Cl₂(2 ml) at room temperature was treated with diethylamine (2 ml). Afterabout 3 hours the solvents were evaporated and the residue was flushedwith CH₂Cl₂ (10 ml). The residue was chromatographed on silica, elutingwith 1) 25:1 and 2) 15:1 CH₂Cl₂/methanol, to give the product as apurple glass (157.4 mg, 82%). ¹H-NMR (CDCl₃) δ 1.15-1.83 (8H, m, CH₂),1.77 (3H, s, MMC CH₃), 2.10 (2H, t, Lys N—CH₂), 2.46 (2H, m, CO—CH₂),2.69 and 3.21 (each 1H, ABq, Phe CH₂), 3.19 (3H, s, MMC O—CH₃),3.20-3.53 (5H, m, GABA N—CH₂, C-1, C-2 and C-3 CH), 3.48 (2H, brs, NH₂),3.68 (2H, m, C-9 CH and Phe CO—CH), 3.76 (3H, s, Mtr O—CH₃), 4.09 and4.82 (each 1H, t and ABq, C-10 CH₂), 4.29 (1H, m, Lys CO—CH), 4.41 (1H,d, C-3 CH), 5.29 (2H, brs, NH₂), 6.60 (1H, brt, GABA NH), 6.79 (2H, d,MeOPh o-CH), 7.10-7.48 (17H, m, Ph), 7.72 (1H, d, amide NH); MS (FAB)967.4 (MH)⁺, 989.2 (M+Na)⁺, 1005.3 (M+K)⁺.

EXAMPLE 76 Preparation of MC-Phe-N^(ε)-Mtr-Lys-GABA-MMC (77)

A solution of Phe-N^(ε)-Mtr-Lys-GABA-MMC (76) (108.9 mg, 0.113 mmoles)in CH₂Cl₂ (15 ml) was added to MC-NHS (0.124 mmoles). The mixture wasstirred at room temperature for 3 days and then the solvent wasevaporated. The residue was chromatographed on silica, eluting with 1)20:1 and 2) 15:1 CH₂Cl₂/methanol, to give the product as a purple glass(75.8 mg, 58%). ¹H-NMR (CDCl₃) δ 1.05-1.90 (14H, m, CH₂), 1.76 (3H, s,MMC CH₃), 2.07 (4H, m, Lys N—CH₂ and caproyl CO—CH₂), 2.49 (2H, m, GABACO—CH₂), 2.98 and 3.20 (each 1H, ABq, Phe CH₂), 3.19 (2H, m, GABAN—CH₂), 3.23 (3H, s, MMC O—CH₃), 3.33 (2H, t, M—CH₂), 3.20-3.53 (3H, m,C-1, C-2 and C-3 CH), 3.68 (1H, ABq, C-9 CH), 3.78 (3H, s, Mtr O—CH₃),4.11 and 4.62 (each 1H, t and ABq, C-10 CH₂), 4.24 (1H, m, Lys CO—CH),4.49 (1H, d, C-3 CH), 5.19 (2H, br, NH₂), 6.27 (1H, d, NH), 6.67 (2H, s,M CH), 6.72 (1H, brt, NH), 6.80 (2H, d, MeOPh o-CH), 7.10-7.47 (17H, m,Ph), 7.19 (1H, d, NH).

EXAMPLE 77 Preparation of MC-Phe-Lys-GABA-MMC . ClCH₂CO₂H (78)

MC-Phe-N^(ε)-Mtr-Lys-GABA-MMC (77) (43.2 mg, 37.2 μmoles) in CH₂Cl₂ (2ml) was treated with anisole (0.405 ml, 100 equiv.) and chloroaceticacid (1M in CH₂Cl₂, 0.40 ml, 11 equiv.). After about 3 hours ether (5ml) was added and the mixture was stored in the freezer for about 1hour. The resulting solid was collected by filtration, washed withether, and triturated with CH₂Cl₂ (36.1 mg, 99%). ¹H-NMR (CDCl₃/CD₃OD) δ1.03-1.82 (8H, m, CH₂), 1.71 (3H, s, MMC CH₃), 2.08 (2H, t, caproylCO—CH₂), 2.40 (2H, brt, GABA CO—CH₂), 2.83 (4H, m, GABA N—CH₂ andN⁺—CH₂), 3.39 (2H, t, M—CH₂), 3.59 (1H, ABq, C-9 CH), 3.95 (1H, t, C-10CH₂), 4.18 (1H, m, Lys CO—CH), 4.42 (1H, d, C-3 CH), 4.67 (2H, m, PheCO—CH and C-10 CH₂), 6.63 (2H, s, M CH), 7.17 (5H, m, Ph); HPLC: (C-18,15 cm column, 65:35 methanol/50 mM triethylammonium formate buffer (pH2.8), 1 ml/min., 360 nm): single peak, retention time: 2.19 min.

EXAMPLE 78 Preparation of Taxol-2′-ethyl carbonate-7-chloroformate (83)

A stirred solution of taxol-2′-ethyl carbonate (82) (154.2 mg, 0.1665mmoles) in CH₂Cl₂ (3 ml) at 0° C. under argon was treated with pyridine(13.5 μl, 1 equiv.) and then diphosgene (10.0 μl, 0.5 equiv.). The icebath was removed and the mixture was stirred at room temperature for onehour and then re-cooled to 0° C. and used immediately.

EXAMPLE 79 Preparation of MC-Phe-N^(ε)-Mtr-Lys-PABC-7-Taxol-2′-ethylcarbonate (84)

A solution of MC-Phe-N^(ε)-Mtr-Lys-PAB-OH (47) (143.9 mg, 0.1665 mmoles)in CH₂Cl₂ (4 ml) was added to the above solution of taxol-2′-ethylcarbonate-7-chloroformate (83) (0.1665 mmoles) at about 0° C. The icebath was removed and the mixture was stirred at room temperature forabout 3 hours. Ethyl acetate was then added and the solution was washedwith pH 5 buffer, water, and brine, dried and evaporated to give acolorless glass which was chromatographed on silica, eluting with 1) 2:1and 2) 1:1 CH₂Cl₂/ethyl acetate, to give the product as a colorlessglass (251.0 mg, 83%). ¹H-NMR (CDCl₃) δ 1.16, 1.21 and 1.78 (each 3H, s,C-16, C-17 and C-19 CH₃), 1.10-1.90 (12H, m, Lys and caproyl CH₂), 1.31(3H, t, ethyl CH₃), 1.91 and 2.60 (each 1H, m, C-6 CH₂), 2.04 (3H, s,C-18 CH₃), 2.12 (4H, t, Lys N—CH₂ and caproyl CO—CH₂), 2.18 and 2.48(each 3H, s, Ac CH₃), 2.22 and 2.40 (each 1H, m, C-14 CH₂), 3.03 (2H, m,Phe CH₂), 3.42 (2H, t, caproyl N—CH₂), 3.97 (1H, d, C-3 CH), 4.29 (2H,m, C-20 CH₂), 4.21 (2H, q, ethyl CH₂), 4.46 and 4.72 (each 1H, m, Pheand Lys CO—CH), 4.96 (1H, d, C-5 CH), 5.16 (2H, q, PAB CH₂), 5.44 (1H,d, C-2′ CH), 5.56 (1H, m, C-7 CH), 5.70 (1H, d, C-2 CH), 5.97 (1H, m,C-3′ CH), 6.26 (1H, m, C-13 CH), 6.40 (1H, s, C-10 CH), 6.65 (2H, s, MCH), 6.78 (2H, d, MeOPh o-CH), 6.98 and 7.60 (each 1H, d, NH), 7.04-8.20(31H, m, Ph), 8.38 (1H, brs, PAB NH); MS (FAB) 1837.2 (M+Na)⁺, 1853.5(M+K)⁺.

EXAMPLE 80 Preparation of MC-Phe-Lys-PABC-7-Taxol-2′-ethylcarbonate.ClCH₂CO₂H (85)

A stirred solution of MC-Phe-N^(ε)-Mtr-Lys-PABC-7-Taxol-2′-ethylcarbonate (84) (80.2 mg, 44.2 μmoles) in CH₂Cl₂ (3.5 ml) at roomtemperature was treated with anisole (0.48 ml, 100 equiv.) andchloroacetic acid (1M in CH₂Cl₂, 0.442 ml, 10 equiv.). After about 3hours ether (15 ml) was added and the mixture was stored in the freezerfor about 2 hours. The resulting white solid was collected by filtrationand washed with ether (72.2 mg, 99%). ¹H-NMR (CDCl₃) δ 1.16, 1.20 and1.80 (each 3H, s, C-16, C-17 and C-19 CH₃), 1.10-1.90 (12H, m, Lys andcaproyl CH₂), 1.30 (3H, t, ethyl CH₃), 1.91 and 2.58 (each 1H, m, C-6CH₂), 2.02 (3H, s, C-18 CH₃), 2.13 (2H, m, caproyl CO—CH₂), 2.17 and2.45 (each 3H, s, Ac CH₃), 2.20 and 2.39 (each 1H, m, C-14 CH₂), 2.97(2H, m, Lys N—CH₂), 3.01 (2H, m, Phe CH₂), 3.42 (2H, t, caproyl N—CH₂),3.97 (1H, d, C-3 CH), 4.29 (4H, m, C-20 CH₂and ethyl CH₂), 4.56 and 4.83(each 1H, m, Phe and Lys CO—CH), 4.95 (1H, d, C-5 CH), 5.17 (2H, q, PABCH₂), 5.42 (1H, d, C-2′ CH), 5.54 (1H, m, C-7 CH), 5.69 (1H, d, C-2 CH),5.97 (1H, m, C-3′ CH), 6.29 (1H, m, C-13 CH), 6.41 (1H, s, C-10 CH),6.66 (2H, s, M CH), 6.98 and 8.39 (each 1H, d, NH), 7.08-8.14 (19H, m,Ph), 9.25 (1H, brs, PAB NH).

EXAMPLE 81 Conjugate Synthesis

A solution (10 ml) of mAb BR96 (10.46 mg/ml, 6.54×10⁻⁵M; concentrationdetermined by UV absorption at 280 nm, 1 mg/ml of mAb equals 1.4 abs.units) in 0.125M potassium phosphate buffer was treated with a freshlyprepared solution (0.523 ml) of 10 mM dithiothreitol (DTT) at about 37°C. for about 3 hours under nitrogen. The solution was transferred to anAmicon cell and was diafiltrated against phosphate buffered saline (PBS)until the effluent was free of SH groups (Ellman reagent). The mAb andSH group concentration was determined (10.11 mg/ml (6.32×10⁻⁵M) and4.48×10⁻⁴M, respectively, representing a molar ratio (MR) of SH to mAbof 7.01). This solution was treated with MC-Phe-Lys-PABC-DOX (5 mg/ml,4.77×10⁻³M) in distilled water (1.2 ml), then left to stand overnight atabout 4° C. The solution was transferred to a dialysis tube and dialyzed3 times against 1 L PBS for about 24 hours at about 4° C. The conjugatesolution was filtered through a MILLEX-GV 0.22 μm filter unit (MilliporeCorp.), and the filtrate was shaken gently for several hours withBio-beads (Bio-Rad Laboratories), followed by another filtration througha MILLIVEXX-GV unit. The concentration of DOX was determined from the UVabsorbance at 495 nm (ε=8030, 283 μm, 164 μg/ml) and that of the mAb at280 nm with a correction for DOX absorbance at 280 nm according to theformula:${{mAb}\quad \left( {{mg}/{ml}} \right)} = \frac{{A280} - \left( {0.724 \times {A495}} \right)}{1.4}$

where A is the observed absorbance at the noted wavelength.

EXAMPLE 82

A solution of Phe-(N^(ε)-MTR)Lys-PABC-DOX in an appropriate solvent istreated with an equivalent amount of N-Succinimidylp-(iodoacetamido)benzoate. The solution is kept at about 30° C. forabout 1 hour and then the solvent is evaporated under reduced pressure.The protecting group MTR is removed from the peptide in the usual mannerand the iodoacetylated peptide is dissolved in water or an organic watermiscible solvent to a known concentration. An appropriate amount of thissolution is added to a solution of thiolated mAb BR96 in PBS to reactwith all thiol groups generated in the mAb. The solution is kept atabout 4° C. for about one hour and then chromatographed over a sizeexclusion column to eliminate low molecular weight compounds from theconjugate. Finally the conjugate solution is shaken with a small amountof Bio-Beads for a few hours, then filtered through a 0.22 micronfilter. The concentration of mAb and DOX is determined from theirabsorption at 280 and 495 nm, respectively and the MR of drug to mAb iscalculated.

EXAMPLE 83

A solution of Phe-(N^(ε)-MTR)Lys-PABC-DOX in an appropriate solvent istreated with an equivalent amount ofN-Succinimidyl-3-(2-pyridynyldithio)-propionate (SPDP). The solution iskept at about 30° C. for about 1 hour and then the solvent is evaporatedunder reduced pressure. The protecting group MTR is removed from thepeptide in the usual manner and the peptide is dissolved in water or anorganic water miscible solvent to a known concentration. An appropriateamount of this solution is added to a solution of thiolated mAb BR96 inPBS to react with all thiol groups generated in the mAb. The solution iskept at about 4° C. for about one hour and then chromatographed over asize exclusion column to eliminate low molecular weight compounds fromthe conjugate. Finally the conjugate solution is shaken with a smallamount of Bio-Beads for a few hours, then filtered through a 0.22 micronfilter. The concentration of mAb and DOX is determined from theirabsorption at 280 and 495 nm, respectively and the MR of drug to mAb iscalculated.

The invention has been described with reference to specific examples,materials and data. As one skilled in the art will appreciate, alternatemeans for using or preparing the various aspects of the invention may beavailable. Such alternate means are to be construed as included withinthe intent and spirit of the present invention as defined by thefollowing claims.

2 4 amino acids amino acid linear peptide unknown 1 Gly Phe Leu Gly 1 4amino acids amino acid linear peptide unknown 2 Ala Leu Ala Leu

We claim:
 1. A compound of the Formula (I):

in which L is a ligand, wherein L is capable of specifically targeting a selected cell population; A is a carboxylic acyl unit; Y is an amino acid; Z is an amino acid; X and W are each a self-immolative spacer; D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, said functional group selected from the group consisting of a primary or secondary amine, hydroxyl, sulflydryl, carboxyl, aldehyde and a ketone; n is an integer of 0 or 1, with the proviso that n cannot be the integer 0 for both Xn and Wn; and m is an integer of 1, 2, 3, 4, 5 or 6; and wherein Y and Z comprise a protein peptide sequence which is selectively enzymatically cleavable by tumor associated proteases.
 2. A compound of claim 1 in which Y is an amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline.
 3. A compound of claim 1 in which Z an amino acid selected from the group consisting of lysine, lysine protected with acetyl or formyl, arginine, arginine protected with tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl, and citrulline.
 4. A compound of claim 1 in which the Y—Z group is phenylalanine-lysine.
 5. A compound of claim 1 in which the Y—Z group is valine-citrulline.
 6. A compound of claim 1 in which the Y—Z group is valine-lysine.
 7. A compound of claim 2 in which Y is phenylalanine.
 8. A compound of claim 2 in which Y is valine.
 9. A compound of claim 3 in which Z is lysine.
 10. A compound of claim 3 in which Z is citrulline.
 11. The compound of claim 1 in which D is a cytotoxic drug.
 12. The compound of claim 2 in which D is a cytotoxic drug.
 13. The compound of claim 3 in which D is a cytotoxic drug.
 14. The compound of claim 4 in which D is a cytotoxic drug.
 15. The compound of claim 5 in which D is a cytotoxic drug.
 16. The compound of claim 6 in which D is a cytotoxic drug.
 17. A compound of claim 1 in which D is an amino containing drug moiety selected from the group consisting of mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N⁸-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, and derivatives thereof.
 18. A compound of claim 2 in which D is an amino containing drug moiety selected from the group consisting of mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N⁸-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, and derivatives thereof.
 19. A compound of claim 3 in which D is an amino containing drug moiety selected from the group consisting of mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N⁸-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, and derivatives thereof.
 20. A compound of claim 4 in which D is an amino containing drug moiety selected from the group consisting of mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N⁸-acetyl spermidine, 1- (2 chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, and derivatives thereof.
 21. A compound of claim 5 in which D is an amino containing drug moiety selected from the group consisting of mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N⁸-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, and derivatives thereof.
 22. A compound of claim 6 in which D is an amino containing drug moiety selected from the group consisting of mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N⁸-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, and derivatives thereof.
 23. A compound of claim 17 in which D is doxorubicin.
 24. A compound of claim 18 in which D is doxorubicin.
 25. A compound of claim 19 in which D is doxorubicin.
 26. A compound of claim 20 in which D is doxorubicin.
 27. A compound of claim 21 in which D is doxorubicin.
 28. A compound of claim 22 in which D is doxorubicin.
 29. A compound of claim 17 in which D is mitomycin-C.
 30. A compound of claim 18 in which D is mitomycin-C.
 31. A compound of claim 19 in which D is mitomycin-C.
 32. A compound of claim 20 in which D is mitomycin-C.
 33. A compound of claim 21 in which D is mitomycin-C.
 34. A compound of claim 22 in which D is mitomycin-C.
 35. A compound of claim 17 in which D is mitomycin-A.
 36. A compound of claim 18 in which D is mitomycin-A.
 37. A compound of claim 19 in which D is mitomycin-A.
 38. A compound of claim 20 in which D is mitomycin-A.
 39. A compound of claim 21 in which D is mitomycin-A.
 40. A compound of claim 22 in which D is mitomycin-A.
 41. A compound of claim 17 in which D is tallysomycin.
 42. A compound of claim 18 in which D is tallysomycin.
 43. A compound of claim 19 in which D is tallysomycin.
 44. A compound of claim 20 in which D is tallysomycin.
 45. A compound of claim 21 in which D is tallysomycin.
 46. A compound of claim 22 in which D is tallysomycin.
 47. A compound of claim 17 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 48. A compound of claim 18 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 49. A compound of claim 19 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 50. A compound of claim 20 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 51. A compound of claim 21 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 52. A compound of claim 22 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 53. A compound of claim 1 in which D is a hydroxyl containing drug moiety selected from the group consisting of etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyne-13-one, anguidine, doxorubicin, morpholino-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, vincristine, vinblastine and derivatives thereof.
 54. A compound of claim 2 in which D is a hydroxyl containing drug moiety selected from the group consisting of etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-ene-2,6-diyne-13-one, anguidine, doxorubicin, morpholino-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, vincristine, vinblastine and derivatives thereof.
 55. A compound of claim 3 in which D is a hydroxyl containing drug moiety selected from the group consisting of etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-ene-2,6-diyne-13-one, anguidine, doxorubicin, morpholino-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, vincristine, vinblastine and derivatives thereof.
 56. A compound of claim 4 in which D is a hydroxyl containing drug moiety selected from the group consisting of etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-ene-2,6-diyne-13-one, anguidine, doxorubicin, morpholino-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, vincristine, vinblastine and derivatives thereof.
 57. A compound of claim 5 in which D is a hydroxyl containing drug moiety selected from the group consisting of etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-ene-2,6-diyne-13-one, anguidine, doxorubicin, morpholino-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, vincristine, vinblastine and derivatives thereof.
 58. A compound of claim 6 in which D is a hydroxyl containing drug moiety selected from the group consisting of etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-ene-2,6-diyne-13-one, anguidine, doxorubicin, morpholino-doxorubicin, and derivatives thereof.
 59. A compound of claim 53 in which D is taxol.
 60. A compound of claim 54 in which D is taxol.
 61. A compound of claim 55 in which D is taxol.
 62. A compound of claim 56 in which D is taxol.
 63. A compound of claim 57 in which D is taxol.
 64. A compound of claim 58 in which D is taxol.
 65. A compound of claim 53 in which D is etoposide.
 66. A compound of claim 54 in which D is etoposide.
 67. A compound of claim 55 in which D is etoposide.
 68. A compound of claim 56 in which D is etoposide.
 69. A compound of claim 57 in which D is etoposide.
 70. A compound of claim 58 in which D is etoposide.
 71. A compound of claim 53 in which D is morpholino-doxorubicin.
 72. A compound of claim 54 in which D is morpholino-doxorubicin.
 73. A compound of claim 55 in which D is morpholino-doxorubicin.
 74. A compound of claim 56 in which D is morpholino-doxorubicin.
 75. A compound of claim 57 in which D is morpholino-doxorubicin.
 76. A compound of claim 58 in which D is morpholino-doxorubicin.
 77. A compound of claim 53 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 78. A compound of claim 54 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 79. A compound of claim 55 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 80. A compound of claim 56 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 81. A compound of claim 57 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 82. A compound of claim 58 in which D is N-(5,5-diacetoxypentyl)doxorubicin.
 83. A compound of claim 1 in which D is a sulfhydryl containing drug moiety selected from the group consisting of esperamicin, 6-mercaptopurine, or derivatives thereof.
 84. A compound of claim 2 in which D is a sulfhydryl containing drug moiety selected from the group consisting of esperamicin, 6-mercaptopurine, or derivatives thereof.
 85. A compound of claim 3 in which D is a sulfhydryl containing drug moiety selected from the group consisting of esperamicin, 6-mercaptopurine, or derivatives thereof.
 86. A compound of claim 4 in which D is a sulfhydryl containing drug moiety selected from the group consisting of esperamicin, 6-mercaptopurine, or derivatives thereof.
 87. A compound of claim 5 in which D is a sulfhydryl containing drug moiety selected from the group consisting of esperamicin, 6-mercaptopurine, or derivatives thereof.
 88. A compound of claim 6 in which D is a sulfhydryl containing drug moiety selected from the group consisting of esperamicin, 6-mercaptopurine, or derivatives thereof.
 89. A compound of claim 1 in which D is a carboxyl containing drug moiety selected from the group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, and derivatives thereof.
 90. A compound of claim 2 in which D is a carboxyl containing drug moiety selected from the group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, and derivatives thereof.
 91. A compound of claim 3 in which D is a carboxyl containing drug moiety selected from the group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, and derivatives thereof.
 92. A compound of claim 4 in which D is a carboxyl containing drug moiety selected from the group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, and derivatives thereof.
 93. A compound of claim 5 in which D is a carboxyl containing drug moiety selected from the group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, and derivatives thereof.
 94. A compound of claim 6 in which D is a carboxyl containing drug moiety selected from the group consisting of methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, and derivatives thereof.
 95. A compound of claim 89 in which D is camptothecin.
 96. A compound of claim 90 in which D is camptothecin.
 97. A compound of claim 91 in which D is camptothecin.
 98. A compound of claim 92 in which D is camptothecin.
 99. A compound of claim 93 in which D is camptothecin.
 100. A compound of claim 94 in which D is camptothecin.
 101. A compound of claim 1 in which D is an aldehyde containing drug.
 102. A compound of claim 2 in which D is an aldehyde containing drug.
 103. A compound of claim 3 in which D is an aldehyde containing drug.
 104. A compound of claim 4 in which D is an aldehyde containing drug.
 105. A compound of claim 5 in which D is an aldehyde containing drug.
 106. A compound of claim 6 in which D is an aldehyde containing drug.
 107. A compound of claim 101 in which D is an anthracycline.
 108. A compound of claim 102 in which D is an anthracycline.
 109. A compound of claim 103 in which D is an anthracycline.
 110. A compound of claim 104 which D is an anthracycline.
 111. A compound of claim 105 in which D is an anthracycline.
 112. A compound of claim 106 in which D is an anthracycline.
 113. A compound of claim 1 in which D is a ketone containing drug.
 114. A compound of claim 2 in which D is a ketone containing drug.
 115. A compound of claim 3 in which D is a ketone containing drug.
 116. A compound of claim 4 in which D is a ketone containing drug.
 117. A compound of claim 5 in which D is a ketone containing drug.
 118. A compound of claim 6 in which D is a ketone containing drug.
 119. A compound of claim 113 in which D is an anthracycline.
 120. A compound of claim 114 in which D is an anthracycline.
 121. A compound of claim 115 in which D is an anthracycline.
 122. A compound of claim 116 in which D is an anthracycline.
 123. A compound of claim 117 in which D is an anthracycline.
 124. A compound of claim 118 in which D is an anthracycline.
 125. A compound of claim 1 in which L is an immunoglobulin, or a fragment thereof.
 126. A compound of claim 2 in which L is an immunoglobulin, or a fragment thereof.
 127. A compound of claim 3 in which L is an immunoglobulin, or a fragment thereof.
 128. A compound of claim 4 in which L is an immunoglobulin, or a fragment thereof.
 129. A compound of claim 5 in which L is an immunoglobulin, or a fragment thereof.
 130. A compound of claim 6 in which L is an immunoglobulin, or a fragment thereof.
 131. A compound of claim 125 in which L is an immunoglobulin selected from the group consisting of BR96, BR64, L6, a reduced BR96, a reduced BR64, a reduced L6, a chimeric BR96, a relaxed chimeric BR64, a chimeric L6, a reduced chimeric BR96, a reduced chimeric BR64, a reduced chimeric L6; and, a fragment thereof.
 132. A compound of claim 126 in which L is an immunoglobulin selected from the group consisting of BR96, BR64, L6, a reduced BR96, a reduced BR64, a reduced L6, a chimeric BR96, a relaxed chimeric BR64, a chimeric L6, a reduced chimeric BR96, a reduced chimeric BR64, a reduced chimeric L6; and, a fragment thereof.
 133. A compound of claim 127 in which L is an immunoglobulin selected from the group consisting of BR96, BR64, L6, a reduced BR96, a reduced BR64, a reduced L6, a chimeric BR96, a relaxed chimeric BR64, a chimeric L6, a reduced chimeric BR96, a reduced chimeric BR64, a reduced chimeric L6; and, a fragment thereof.
 134. A compound of claim 128 in which L is an immunoglobulin selected from the group consisting of BR96, BR64, L6, a reduced BR96, a reduced BR64, a reduced L6, a chimeric BR96, a relaxed chimeric BR64, a chimeric L6, a reduced chimeric BR96, a reduced chimeric BR64, a reduced chimeric L6; and, a fragment thereof.
 135. A compound of claim 129 in which L is an immunoglobulin selected from the group consisting of BR96, BR64, L6, a reduced BR96, a reduced BR64, a reduced L6, a chimeric BR96, a relaxed chimeric BR64, a chimeric L6, a reduced chimeric BR96, a reduced chimeric BR64, a reduced chimeric L6; and, a fragment thereof.
 136. A compound of claim 130 in which L is an immunoglobulin selected from the group consisting of BR96, BR64, L6, a reduced BR96, a reduced BR64, a reduced L6, a chimeric BR96, a relaxed chimeric BR64, a chimeric L6, a reduced chimeric BR96, a reduced chimeric BR64, a reduced chimeric L6; and, a fragment thereof.
 137. A compound of claim 131 in which L is a chimeric BR96, a reduced chimeric BR96; or a fragment thereof.
 138. A compound of claim 132 in which L is a chimeric BR96, a reduced chimeric BR96; or a fragment thereof.
 139. A compound of claim 133 in which L is a chimeric BR96, a reduced chimeric BR96; or a fragment thereof.
 140. A compound of claim 134 in which L is a chimeric BR96, a reduced chimeric BR96; or a fragment thereof.
 141. A compound of claim 135 in which L is a chimeric BR96, a reduced chimeric BR96; or a fragment thereof.
 142. A compound of claim 136 in which L is a chimeric BR96, a reduced chimeric BR96; or a fragment thereof.
 143. A compound of claim 1 in which L is a ligand selected from the group consisting of bombesin, EDG, transferrin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, TFG-α, TFG-β, VGF, insulin and insulin-like growth factors I and II.
 144. A compound of claim 2 in which L is a ligand selected from the group consisting of bombesin, EDG, transferrin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, TFG-α, TFG-β, VGF, insulin and insulin-like growth factors I and II.
 145. A compound of claim 3 in which L is a ligand selected from the group consisting of bombesin, EDG, transferrin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, TFG-α, TFG-β, VGF, insulin and insulin-like growth factors I and II.
 146. A compound of claim 4 in which L is a ligand selected from the group consisting of bombesin, EDG, transferrin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, TFG-α, TFG-β, VGF, insulin and insulin-like growth factors I and II.
 147. A compound of claim 5 in which L is a ligand selected from the group consisting of bombesin, EDG, transferrin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, TFG-α, TFG-β, VGF, insulin and insulin-like growth factors I and II.
 148. A compound of claim 6 in which L is a ligand selected from the group consisting of bombesin, EDG, transferrin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, TFG-α, TFG-β, VGF, insulin and insulin-like growth factors I and II.
 149. A compound of claim 1 in which L is a ligand selected from the group consisting of carbohydrates, lectins, and apoprotein from low density lipoproteins.
 150. A compound of claim 2 in which L is a ligand selected from the group consisting of carbohydrates, lectins, and apoprotein from low density lipoproteins.
 151. A compound of claim 3 in which L is a ligand selected from the group consisting of carbohydrates, lectins, and apoprotein from low density lipoproteins.
 152. A compound of claim 4 in which L is a ligand selected from the group consisting of carbohydrates, lectins, and apoprotein from low density lipoproteins.
 153. A compound of claim 5 in which L is a ligand selected from the group consisting of carbohydrates, lectins, and apoprotein from low density lipoproteins.
 154. A compound of claim 6 in which L is a ligand selected from the group consisting of carbohydrates, lectins, and apoprotein from low density lipoproteins.
 155. A compound of claim 1 in which X is the compound having the Formula

in which T is O, N, or S.
 156. A compound of claim 2 in which X is the compound having the Formula

in which T is O, N, or S.
 157. A compound of claim 3 in which X is the compound having the Formula

in which T is O, N, or S.
 158. A compound of claim 4 in which X is the compound having the Formula

in which T is O, N, or S.
 159. A compound of claim 5 in which X is the compound having the Formula

in which T is O, N, or S.
 160. A compound of claim 6 in which X is the compound having the Formula

in which T is O, N, or S.
 161. A compound of claim 155 in which X is p-aminobenzyl-carbamoyloxy.
 162. A compound of claim 156 in which X is p-aminobenzyl-carbamoyloxy.
 163. A compound of claim 157 in which X is p-aminobenzyl-carbamoyloxy.
 164. A compound of claim 158 in which X is p-aminobenzyl-carbamoyloxy.
 165. A compound of claim 159 in which X is p-aminobenzyl-carbamoyloxy.
 166. A compound of claim 160 in which X is p-aminobenzyl-carbamoyloxy.
 167. A compound of claim 1 in which X is the compound having the Formula —HN—R¹—COT in which R¹ is C₁-C₅ alkyl, T is O, N or S.
 168. A compound of claim 2 in which X is the compound having the Formula —HN—R¹—COT in which R¹ is C₁-C₅ alkyl, T is O, N or S.
 169. A compound of claim 3 in which X is the compound having the Formula —HN—R¹—COT in which R¹ is C₁-C₅ alkyl, T is O, N or S.
 170. A compound of claim 4 in which X is the compound having the Formula —HN—R¹—COT in which R¹ is C₁-C₅ alkyl, T is O, N or S.
 171. A compound of claim 5 in which X is the compound having the Formula —HN—R¹—COT in which R¹ is C₁-C₅ alkyl, T is O, N or S.
 172. A compound of claim 6 in which X is the compound having the Formula —HN—R¹—COT in which R¹ is C₁-C₅ alkyl, T is O, N or S.
 173. A compound of claim 167 in which X is γ-aminobutyric acid.
 174. A compound of claim 168 in which X is γ-aminobutyric acid.
 175. A compound of claim 169 in which X is γ-aminobutyric acid.
 176. A compound of claim 170 in which X is γ-aminobutyric acid.
 177. A compound of claim 171 in which X is γ-aminobutyric acid.
 178. A compound of claim 172 in which X is γ-aminobutyric acid.
 179. A compound of claim 167 in which X is α,α-dimethyl γ-aminobutyric acid.
 180. A compound of claim 168 in which X is α,α-dimethyl γ-aminobutyric acid.
 181. A compound of claim 169 in which X is α,α-dimethyl γ-aminobutyric acid.
 182. A compound of claim 170 in which X is α,α-dimethyl γ-aminobutyric acid.
 183. A compound of claim 171 in which X is α,α-dimethyl γ-aminobutyric acid.
 184. A compound of claim 172 in which X is α,α-dimethyl γ-aminobutyric acid.
 185. A compound of claim 167 in which X is β,β-dimethyl γ-aminobutyric acid.
 186. A compound of claim 168 in which X is β,β-dimethyl γ-aminobutyric acid.
 187. A compound of claim 169 in which X is β,β-dimethyl γ-aminobutyric acid.
 188. A compound of claim 170 in which X is β,β-dimethyl γ-aminobutyric acid.
 189. A compound of claim 171 in which X is β,β-dimethyl γ-aminobutyric acid.
 190. A compound of claim 172 in which X is β,β-dimethyl γ-aminobutyric acid.
 191. A compound of claim 1 in which X is the compound having the Formula

in which T is O, N, or S, R² is H or C₁-C₅ alkyl.
 192. A compound of claim 2 in which X is the compound having the Formula

in which T is O, N, or S, R² is H or C₁-C₅ alkyl.
 193. A compound of claim 3 in which X is the compound having the Formula

in which T is O, N, or S, R² is H or C₁-C₅ alkyl.
 194. A compound of claim 4 in which X is the compound having the Formula

in which T is O, N, or S, R² is H or C₁-C₅ alkyl.
 195. A compound of claim 5 in which X is the compound having the Formula

in which T is O, N, or S, R² is H or C₁-C₅ alkyl.
 196. A compound of claim 6 in which X is the compound having the Formula

in which T is O, N, or S, R² is H or C₁-C₅ alkyl.
 197. A compound of claim 1 in which W is

in which T is O, S or N.
 198. A compound of claim 2 in which W is

in which T is O, S or N.
 199. A compound of claim 3 in which W is

in which T is O, S or N.
 200. A compound of claim 4 in which W is

in which T is O, S or N.
 201. A compound of claim 5 in which W is

in which T is O, S or N.
 202. A compound of claim 6 in which W is

in which T is O, S or N.
 203. A compound of claim 1 in which A is

in which q is 1-10.
 204. A compound of claim 2 in which A is

in which q is 1-10.
 205. A compound of claim 3 in which A is

in which q is 1-10.
 206. A compound of claim 4 in which A is

in which q is 1-10.
 207. A compound of claim 5 in which A is

in which q is 1-10.
 208. A compound of claim 6 in which A is

in which q is 1-10.
 209. A compound of claim 1 in which A is 4-(N-succinimidomethyl)cyclohexane-1-carbonyl.
 210. A compound of claim 2 in which A is 4-(N-succinimidomethyl)cyclohexane-1-carbonyl.
 211. A compound of claim 3 in which A is 4-(N-succinimidomethyl)cyclohexane-1-carbonyl.
 212. A compound of claim 4 in which A is 4-(N-succinimidomethyl)cyclohexane-1-carbonyl.
 213. A compound of claim 5 in which A is 4-(N-succinimidomethyl)cyclohexane-1-carbonyl.
 214. A compound of claim 6 in which A is 4-(N-succinimidomethyl)cyclohexane-1-carbonyl.
 215. A compound of claim 1 in which A is m-succinimidobenzoyl.
 216. A compound of claim 2 in which A is m-succinimidobenzoyl.
 217. A compound of claim 3 in which A is m-succinimidobenzoyl.
 218. A compound of claim 4 in which A is m-succinimidobenzoyl.
 219. A compound of claim 5 in which A is m-succinimidobenzoyl.
 220. A compound of claim 6 in which A is m-succinimidobenzoyl.
 221. A compound of claim 1 in which A is 4-(p-succinimidophenyl)butyryl.
 222. A compound of claim 2 in which A is 4-(p-succinimidophenyl)butyryl.
 223. A compound of claim 3 in which A is 4-(p-succinimidophenyl)butyryl.
 224. A compound of claim 4 in which A is 4-(p-succinimidophenyl)butyryl.
 225. A compound of claim 5 in which A is 4-(p-succinimidophenyl)butyryl.
 226. A compound of claim 6 in which A is 4-(p-succinimidophenyl)butyryl.
 227. A compound of claim 1 in which A is 4-(2-acetamido)benzoyl.
 228. A compound of claim 2 in which A is 4-(2-acetamido)benzoyl.
 229. A compound of claim 3 in which A is 4-(2-acetamido)benzoyl.
 230. A compound of claim 4 in which A is 4-(2-acetamido)benzoyl.
 231. A compound of claim 5 in which A is 4-(2-acetamido)benzoyl.
 232. A compound of claim 6 in which A is 4-(2-acetamido)benzoyl.
 233. A compound of claim 1 in which A is 3-thiopropionyl.
 234. A compound of claim 2 in which A is 3-thiopropionyl.
 235. A compound of claim 3 in which A is 3-thiopropionyl.
 236. A compound of claim 4 in which A is 3-thiopropionyl.
 237. A compound of claim 5 in which A is 3-thiopropionyl.
 238. A compound of claim 6 in which A is 3-thiopropionyl.
 239. A compound of claim 1 in which A is 4-(1-thioethyl)-benzoyl.
 240. A compound of claim 2 in which A is 4-(1-thioethyl)-benzoyl.
 241. A compound of claim 3 in which A is 4-(1-thioethyl)-benzoyl.
 242. A compound of claim 4 in which A is 4-(1-thioethyl)-benzoyl.
 243. A compound of claim 5 in which A is 4-(1-thioethyl)-benzoyl.
 244. A compound of claim 6 in which A is 4-(1-thioethyl)-benzoyl.
 245. A compound of claim 1 in which A is 6-(3-thiopropionylamido)-hexanoyl.
 246. A compound of claim 2 in which A is 6-(3-thiopropionylamido)-hexanoyl.
 247. A compound of claim 3 in which A is 6-(3-thiopropionylamido)-hexanoyl.
 248. A compound of claim 4 in which A is 6-(3-thiopropionylamido)-hexanoyl.
 249. A compound of claim 5 in which A is 6-(3-thiopropionylamido)-hexanoyl.
 250. A compound of claim 6 in which A is 6-(3-thiopropionylamido)-hexanoyl.
 251. A compound of claim 1 which is BR96-succinimidocaproyl-phenylalanine-lysine-p-aminobenzyl-carbamoyloxy-doxrubicin.
 252. A compound of claim 1 which is BR96-succinimidocaproyl-valine-lysine-p-aminobenzyl-carbamoyloxy-doxorubicin.
 253. A compound of claim 1 which is BR96-succinimidocaproyl-valine-citrulline-p-aminobenzyl-carbamoyloxy-doxorubicin.
 254. A compound of claim 1 which is BR96-succinimidocaproyl-phenylalanine-lysine-p-aminobenzyl-carbamoyloxy-2′-taxol.
 255. A compound of claim 1 which is BR96-succinimidocaproyl-phenylalanine-lysine-p-aminobenzyl-carbamoyloxy-7-taxol.
 256. A compound of claim 1 which is BR96-succinimidocaproyl-phenylalanine-lysine-p-aminobenzyl-carbamoyloxy-mitomycin-C.
 257. A compound of claim 1 which is BR96-succinimidocaproyl-phenylalanine-lysine-gamma-aminobutyric acid-mitomycin-C.
 258. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier. 